Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy

ABSTRACT

The present invention provides compositions and methods for regulating the specificity and activity of T cells. In one embodiment, the invention provides a type of chimeric antigen receptor (CAR) wherein the CAR is termed a “KIR-CAR” which is a CAR design comprising a component of a receptor naturally found on natural killer (NK) cells. In one embodiment, the NK receptor includes but is not limited to a naturally occurring activating and inhibitory receptor of NK cells known as a killer cell immunoglobulin-like receptor (KIR).

This application is a divisional of U.S. Ser. No. 14/214,824, filed Mar.15, 2014, which claims priority to U.S. Ser. No. 61/793,443 filed Mar.15, 2013, the entire contents which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under PN2 EY016586awarded by the National Institutes of Health (NIH). The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 10, 2014, isnamed N2067-704910_SL.txt and is 246,698 bytes in size.

BACKGROUND OF THE INVENTION

With the use of gene transfer technologies, T cells can be geneticallymodified to stably express antibody binding domains on their surfacethat endow the T cells with specificities that are independent of theconstraints imposed by the major histocompatibility complex (MHC).Chimeric antigen receptors (CARs) represent synthetic proteins expressedon T-cells (CART cells) that fuse an antigen recognition fragment of anantibody (e.g., an scFv, or single-chain variable region fragment) withan intracellular domain of the CD3-zeta chain. Upon interaction with atarget cell expressing the scFv's cognate antigen, CARs expressed on Tcell cells can trigger T-cell activation leading to target cell killing(also referred to as target cell lysis). When combined with additionalcostimulatory signals such as the intracellular domain of CD137 or CD28,these receptors are also capable of generating proliferation. However,some of this proliferation appears to be antigen-independent, unlikenormal T cell receptor (TCR) responses (Milone et al., 2009, Mol Ther17(8):1453-64). Artificial receptors do not fully reproduce theintracellular signal transduction produced by natural TCR binding toantigenic peptide complexed with MHC molecules (Brocker, 2000, Blood96(5):1999-2001). The signaling defects may limit the long-term survivalof CART cells upon adoptive transfer in the absence of high levels ofcytokines like IL-2 (Lo et al., 2010, Clin Cancer Res 16(10):2769-80).They also have altered regulation that might be beneficial in someanti-cancer applications (Loskog et al., 2006, Leukemia 20(10):1819-28),but these regulatory defects also lead to potential challenges tocontrolling their “off-target” activity against normal tissues that alsoexpress antigen, even at extremely low levels. These “off-target”effects are a serious limitation to CAR-based therapeutics, and haveresulted in probable deaths during early Phase I evaluation ofCAR-modified T cells (Morgan et al., 2010, Mol Ther 18(4):843-51).

Thus, there is a need in the art for alternative approaches forconstructing CARs that overcome the limitations to current CAR-basedtherapeutics. The present invention addresses this unmet need in theart.

SUMMARY

In a first aspect, the invention features a purified, or non-naturallyoccurring, NKR-CAR comprising one, two or all of an extra-cellularantigen binding domain, a transmembrane domain, e.g., an NKRtransmembrane domain, and a cytoplasmic domain, e.g., an NKR cytoplasmicdomain.

In one embodiment, said NKR-CAR comprises an extra-cellular antigenbinding domain; a transmembrane domain and an NKR cytoplasmic domain. Inone embodiment, said NKR-CAR comprises a KIR-CAR, e.g., an actKIR-CAR orinhKIR-CAR, a NCR-CAR, e.g., an actNCR-CAR, a SLAMF-CAR, e.g., aninhSLAMF-CAR, a FcR-CAR, e.g., CD16-CAR, e.g., an actCD16-CAR, orCD64-CAR, e.g., an actCD64-CAR, or a Ly49-CAR, e.g., an actLy49-CAR orinhLy49-CAR. In one embodiment, the NKR-CAR comprises a transmembranedomain and an extra-cellular antigen binding domain, and furthercomprising a hinge domain disposed between said transmembrane domain andsaid extra-cellular antigen binding domain.

In another aspect, the invention features a nucleic acid, e.g., apurified or non-naturally occurring, nucleic acid, e.g., a nucleic acidcomprising a DNA, or RNA, sequence, e.g., a mRNA, comprising a sequencethat encodes a NKR-CAR described herein. In one embodiment, the nucleicacid further comprising a sequence that encodes an adaptor molecule orintracellular signaling domain that interacts with said NKR-CAR.

In another aspect, the invention features a cytotoxic cell, e.g., anaturally or non-naturally occurring T cell, NK cell or cytotoxic T cellor NK cell line comprising a NKR-CAR described herein. In oneembodiment, the cytotoxic cell further comprises an adaptor molecule orintracellular signaling domain that interacts with said NKR-CAR.

In another aspect, the invention features a method of making a cytotoxiccell, e.g., a naturally or non-naturally occurring T cell, NK cell orcytotoxic T cell or NK cell line comprising a NKR-CAR, described hereincomprising introducing into a cytotoxic cell a nucleic acid, e.g., amRNA, comprising a sequence that encodes a NKR-CAR, described herein. Inone embodiment, the method further comprises making a NKR-CAR, describedherein, in the cytotoxic cell.

In another aspect, the invention features a method of treating asubject, e.g., a method of providing an anti-tumor immunity in a mammal,comprising administering to the mammal an effective amount of acytotoxic cell, e.g., a naturally or non-naturally occurring T cell, NKcell or cytotoxic T cell or NK cell line comprising a NKR-CAR describedherein.

In another aspect, the invention features a purified, or non-naturallyoccurring, KIR-CAR comprising an extra-cellular antigen binding domainand a transmembrane domain, e.g., a KIR transmembrane domain, orcytoplasmic domain, e.g., an ITIM-containing cytoplasmic domain, or aKIR-cytoplasmic domain. In one embodiment, the KIR-CAR comprises anextra-cellular antigen binding domain, a transmembrane domain, and anITIM-containing cytoplasmic domain, or a KIR-cytoplasmic domain.

In one embodiment, said transmembrane domain can interact with, e.g.,bind, the transmembrane domain of DAP12. In one embodiment, saidtransmembrane domain comprises a positively charged moiety, e.g., anamino acid residue comprising a positively charged moiety, e.g., sidechain. In one embodiment, said transmembrane domain comprises aKIR-transmembrane domain.

In one embodiment, said KIR-CAR is an activating KIR-CAR. In oneembodiment, said KIR-CAR comprises a KIR-transmembrane domain. In oneembodiment, said KIR-CAR is an inhibitory KIR-CAR. In one embodiment,said KIR-CAR comprises a KIR-cytoplasmic domain. In one embodiment, saidKIR-CAR comprises an extra-cellular antigen binding domain and atransmembrane domain, e.g., a transmembrane domain comprising apositively charged moiety, e.g., an amino acid residue comprising apositively charged moiety, e.g., side chain, or a KIR-transmembranedomain.

In one embodiment, a KIR-CAR described herein comprises an antigenbinding domain comprising an scFv. In one embodiment, said antigenbinding domain comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence, or a non-antibody scaffold, e.g., a fibronectin, e.g., afibronectin type III antibody-like molecule. In one embodiment, saidantigen binding domain comprises a nanobody. In one embodiment, saidantigen binding domain comprises a camelid VHH domain.

In one embodiment, a KIR-CAR described herein comprises an extracellularhinge domain. In one embodiment, the extracellular hinge domain is otherthan a KIR hinge domain, e.g., other than a KIR2DS2 hinge domain. In oneembodiment, the extracellular hinge domain is derived from a naturalmolecule. In one embodiment, the extracellular hinge domain is derivedfrom a natural molecule other than a KIR. In one embodiment, theextracellular hinge domain comprises a non-naturally occurringpolypeptide sequence. In one embodiment, the extracellular hinge domaincomprises the extracellular hinge from human CD8-alpha. In oneembodiment, the extracellular hinge domain comprises a syntheticextracellular hinge. In one embodiment, the extracellular hinge domainis less than 50, 20, or 10 amino acids in length. In one embodiment, theextracellular hinge domain has fewer amino acids than a KIR2DS2 hingedomain.

In one embodiment, the KIR-CAR described herein is an actKIR-CAR. In oneembodiment, said actKIR-CAR comprises a transmembrane domain comprisinga positively charged moiety, e.g., an amino acid residue comprising apositively charged moiety, e.g., a positively charged side chain or anactKIR transmembrane domain. In one embodiment, said actKIR-CAR caninteract with and promote signaling from an ITAM-containing polypeptideor adaptor molecule. In one embodiment, said actKIR-CAR can interactwith and promote signaling from a DAP12 polypeptide. In one embodiment,said actKIR-CAR comprises a KIR D domain. In one embodiment, saidactKIR-CAR comprises a KIR D1 domain. In one embodiment, said actKIR-CARcomprises a KIR D2 domain. In one embodiment, said actKIR-CAR said actKIR-CAR does not comprise a KIR D domain. In one embodiment, saidactKIR-CAR comprises a KIR2DS2 transmembrane domain. In one embodiment,said actKIR-CAR further comprises a KIR2DS2 cytoplasmic domain. In oneembodiment, said actKIR-CAR does not comprise a KIR D domain.

In one embodiment, the antigen binding domain of a KIR-CAR describedherein binds an antigen present on a target cell, e.g., a cancer cell.In one embodiment, said antigen binding domain binds an antigen that ismore highly expressed on a target cell, e.g., a cancer cell, than anon-target cell, e.g., a non-cancerous cell, e.g., a non cancerous cellof the same type as the target cell. In one embodiment, said antigenbinding domain is binds an antigen described herein.

In one embodiment, the KIR-CAR described herein is an inhKIR-CAR. In oneembodiment, the inhKIR-CAR comprises an inhKIR transmembrane domain. Inone embodiment, the inhKIR-CAR inhKIR-CAR comprises an ITIM-containingcytoplasmic domain, e.g., an inhKIR cytoplasmic domain, e.g., a KIR2DLor KIR3DL cytoplasmic domain. In one embodiment, the inhKIR-CARcomprises a transmembrane other than a KIR transmembrane, e.g., atransmembrane domain from PD-1, CTLA4 or ITIM-containing receptors fromILT (CD85), Siglec, LMIR (CD300) and/or SLAM gene families of receptors.In one embodiment, the inhKIR-CAR comprises a cytoplasmic domain from aninhibitory receptor other than a KIR, e.g., from PD-1, CTLA4 orITIM-containing receptors from ILT (CD85), Siglec, LMIR (CD300) and/orSLAM gene families of receptors. In one embodiment, the inhKIR-CARcomprises a transmembrane and cytoplasmic domain from an inhibitoryreceptor other than a KIR, e.g., transmembrane and cytoplasmic domain,independently, from e.g., PD-1, CTLA4 or ITIM-containing receptors fromILT (CD85), Siglec, LMIR (CD300) and/or SLAM gene families of receptors.In one embodiment, said cytoplasmic domain comprises an ITIM. In oneembodiment, the inhKIR-CAR comprises a KIR D domain. In one embodiment,the inhKIR-CAR comprises a KIR D0 domain. In one embodiment, theinhKIR-CAR comprises a KIR D1 domain. In one embodiment, the inhKIR-CARcomprises a KIR D2 domain. In one embodiment, the inhKIR-CAR does notcomprise a KIR D domain.

In one embodiment, the antigen binding domain of the inhKIR-CARsdescribed herein binds an antigen not present on a target cell, e.g., acancer cell. In one embodiment, said antigen binding domain binds anantigen that is more highly expressed on a non-target cell, e.g., anon-cancer cell, than a target cell, e.g., cancerous cell, e.g., acancerous cell of the same type as the target cell. In one embodiment,said antigen binding domain binds desmoglein1/3 (DSG1/3). In anembodiment, an inhCAR, e.g., an inhTCAR or inhNKR-CAR, e.g., aninhKIR-CAR, and an actCAR, e.g., an actTCAR or actNKR-CAR, e.g., anactKIR-CAR, are provided in which the inhCAR comprises an antigenbinding domain that targets desmoglein1/3 (DSG1/3) and the actCARcomprises an antigen binding domain that targets an antigen other thanDSG1/3, e.g., EGFR. In an embodiment, this pair is used to treat an EGFRexpressing cancer, e.g., an adenocarcinoma of the lung or colon. In anembodiment the cancer cells express less DSG1/3 than non-cancer cells.In an embodiment this combination can minimize CAR-mediated attack ofskin cells or squamous cells of the GI track (i.e. oral mucosa). In oneembodiment, said antigen binding domain binds an ephrin receptor or aclaudin.

In another aspect, the invention features a nucleic acid, e.g., apurified or non-naturally occurring, nucleic acid, e.g., a nucleic acidcomprising a DNA, or RNA, sequence, e.g., a mRNA, comprising (a) asequence that encodes a KIR-CAR, e.g., a first KIR-CAR described herein.In one embodiment, said KIR-CAR, e.g., said first KIR-CAR, is anactKIR-CAR, e.g., an actKIR-CAR described herein. In one embodiment,said KIR-CAR, e.g., said first KIR-CAR, is an inhKIR-CAR, e.g., aninhKIR-CAR described herein. In one embodiment, said nucleic acidcomprises a DNA sequence. In one embodiment, said nucleic acid comprisesa RNA sequence, e.g., a mRNA sequence.

In an embodiment, said nucleic acid comprises sequence that encodes aKIR-CAR, e.g., an actKIR-CAR, and sequence that encodes an inhibitorymolecule comprising: an inhKIR cytoplasmic domain; a transmembranedomain, e.g., a KIR transmembrane domain; and an inhibitor cytoplasmicdomain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. In anembodiment the inhibitory molecule is a naturally occurring inhKIR, or asequence sharing at least 50, 60, 70, 80, 85, 90, 95, or 99% homologywith, or that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 residues from, a naturally occurring inhKIR.

In an embodiment, said nucleic acid comprises sequence that encodes aKIR-CAR, e.g., an actKIR-CAR, and sequence that encodes an inhibitorymolecule comprising: a SLAM family cytoplasmic domain; a transmembranedomain, e.g., a SLAM family transmembrane domain; and an inhibitorcytoplasmic domain, e.g., a SLAM family domain, e.g., an SLAM familyITIM domain. In an embodiment the inhibitory molecule is a naturallyoccurring SLAM family member, or a sequence sharing at least 50, 60, 70,80, 85, 90, 95, or 99% homology with, or that differs by no more than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues from, a naturallyoccurring SLAM family member.

In one embodiment, said nucleic acid described herein further comprises(b) a sequence that encodes a second KIR-CAR described herein, e.g., asecond KIR-CAR that is different from said first KIR-CAR. In oneembodiment, (a) and (b) are disposed on the same nucleic acid molecule,e.g., the same vector, e.g., the same viral vector, e.g., a lenti-viralvector. In one embodiment, one of (a) and (b) is disposed on a firstnucleic acid molecule, e.g., a first vector, e.g., a viral vector, e.g.,a lenti-viral vector, and the other is disposed on a second nucleic acidmolecule, e.g., a second vector, e.g., a viral vector, e.g., alenti-viral vector. In one embodiment, said first KIR-CAR and saidsecond KIR-CAR is an actKIR-CAR. In an embodiment, engagement of eitheract KIR-CAR alone is insufficient to trigger substantial levels ofactivation. In an embodiment, engagement of both the first and secondactKIR-CAR gives an additive, or synergistic, level of activation. Inone embodiment, said first KIR-CAR and said second KIR-CAR is aninhKIR-CAR. In one embodiment, one of said first KIR-CAR and said secondKIR-CAR is an actKIR-CAR and the other is an inhKIR-CAR. In oneembodiment, said actKIR-CAR is an actKIR-CAR described herein. In oneembodiment, said inhKIR-CAR is an inhKIR-CAR described herein. In oneembodiment, the nucleic acid described herein comprises an actKIR-CARdescribed herein and an inhKIR-CAR described herein.

In an embodiment the nucleic further comprises (c) sequence that encodesan intracellular signaling domain, e.g., an adaptor molecule, which canproduce an activating signal. In one embodiment, said intracellularsignaling domain comprises an ITAM motif. In one embodiment, saidsequence encodes a DAP 12 polypeptide comprising a DAP 12 intracellularsignaling domain. In one embodiment, said DAP 12 polypeptide furthercomprises a transmembrane domain. In one embodiment, said DAP 12polypeptide further comprises an extracellular domain. In oneembodiment, each of (a), (b), and (c) are present on the same nucleicacid molecule, e.g., a vector, e.g., a viral vector, e.g., a lenti-viralvector. In one embodiment, one of (a), (b), and (c) is encoded on afirst nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g.,a lenti-viral vector and a second and third of (a), (b), and (c) isencoded on a second nucleic acid molecule, e.g., a vector, e.g., a viralvector, e.g., a lenti-viral vector. In one embodiment (a) is present ona first nucleic acid molecule, e.g., a vector, e.g., a viral vector,e.g., a lenti-viral vector, and (b) and (c) are present on a secondnucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., alenti-viral vector. In another embodiment, (b) is present on a firstnucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., alenti-viral vector, and (a) and (c) are present on a second nucleic acidmolecule, e.g., a vector, e.g., a viral vector, e.g., a lenti-viralvector. In one embodiment, (c) is present on a first nucleic acidmolecule, e.g., a vector, e.g., a viral vector, e.g., a lenti-viralvector, and (b) and (a) are present on a second nucleic acid molecule,e.g., a vector, e.g., a viral vector, e.g., a lenti-viral vector. In oneembodiment, each of (a), (b), and (c) are present on different nucleicacid molecules, e.g., different vectors, e.g., viral vectors, e.g., alenti-viral vectors.

In an embodiment, (i) the antigen binding domain of one of said firstKIR-CAR said second KIR-CAR does not comprise a light chain variabledomain and a heavy chain variable domain, (ii) the antigen bindingdomain of one of said first KIR-CAR said second KIR-CAR is an scFv, andthe other is other is other than an scFv, (iii) when present on thesurface of a cell, the antigen binding domains of said first KIR-CAR andsaid second KIR-CAR, associate with one another less than if both werescFv antigen binding domains, (iv) wherein, when present on the surfaceof a cell, binding of the antigen binding domain of said first KIR-CARto its cognate antigen is not substantially reduced by the presence ofsaid second KIR-CAR, (v) the antigen binding domain of one of said firstKIR-CAR said second KIR-CAR, comprises a single VH domain, e.g., acamelid, shark, or lamprey single VH domain, or a single VH domainderived from a human or mouse sequence, or a non-antibody scaffold,e.g., a fibronectin, e.g., a fibronectin type III antibody-likemolecule, (vi) the antigen binding domain of one of said first KIR-CARsaid second KIR-CAR, is an scFv, and the other comprises a single VHdomain, e.g., a camelid, shark, or lamprey single VH domain, or a singleVH domain derived from a human or mouse sequence or a non-antibodyscaffold, e.g., a fibronectin, e.g., a fibronectin type IIIantibody-like molecule, (vii) the antigen binding domain of one of saidfirst KIR-CAR said second KIR-CAR, is an scFv, and the other comprises ananobody, or (viii) the antigen binding domain of one of said firstKIR-CAR said second KIR-CAR, is an scFv, and the other comprises acamelid VHH domain.

In one embodiment, the antigen binding domain of one of said firstKIR-CAR said second KIR-CAR does not comprise a light chain variabledomain and a heavy chain variable domain. In one embodiment, the antigenbinding domain of one of said first KIR-CAR said second KIR-CAR is anscFv, and the other is other than an scFv. In one embodiment, whenpresent on the surface of a cell, the antigen binding domains of saidfirst KIR-CAR said second KIR-CAR, associate with one another less thanif both were scFv antigen binding domains. In one embodiment, whenpresent on the surface of a cell, binding of the antigen binding domainof said first KIR-CAR to its cognate antigen is not substantiallyreduced by the presence of said second KIR-CAR. In one embodiment, theantigen binding domain of one of said first KIR-CAR said second KIR-CAR,comprises a single VH domain, e.g., a camelid, shark, or lamprey singleVH domain, or a single VH domain derived from a human or mouse sequenceor a non-antibody scaffold, e.g., a fibronectin, e.g., a fibronectintype III antibody-like molecule. In one embodiment, the antigen bindingdomain of one of said first KIR-CAR said second KIR-CAR, is an scFv, andthe other comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence or a non-antibody scaffold, e.g., a fibronectin, e.g., afibronectin type III antibody-like molecule. In one embodiment, theantigen binding domain of one of said first KIR-CAR said second KIR-CAR,is an scFv, and the other comprises a nanobody. In one embodiment, theantigen binding domain of one of said first KIR-CAR said second KIR-CAR,is an scFv, and the other comprises a camelid VHH domain.

In a embodiment the nucleic acid comprises a sequence that encodes aTCAR. In one embodiment, said TCAR comprises an antigen binding domainand an activating cytoplasmic domain from the T cell receptor complexwith CD3 e.g. CD3 zeta chain, CD3 epsilon chain, CD3 gamma chain, CD3delta chain. In one embodiment, said TCAR comprises a costimulatorydomain from costimulatory receptor e.g. CD28, CD137, CD27, ICOS or OX40.

In an embodiment (i) the antigen binding domain of one of said KIR-CARsaid TCAR does not comprise a light chain variable domain and a heavychain variable domain, (ii) the antigen binding domain of one of saidKIR-CAR said TCAR is an scFv, and the other is other than an scFv, (iii)when present on the surface of a cell, the antigen binding domains ofsaid KIR-CAR and said TCAR, associate with one another less than if bothwere scFv antigen binding domains, (iv) when present on the surface of acell, binding of the antigen binding domain of said KIR-CAR to itscognate antigen is not substantially reduced by the presence of saidsecond TCAR, (v) the antigen binding domain of one of said KIR-CAR saidTCAR, comprises a single VH domain, e.g., a camelid, shark, or lampreysingle VH domain, or a single VH domain derived from a human or mousesequence or a non-antibody scaffold, e.g., a fibronectin, e.g., afibronectin type III antibody-like molecule, (vi) the antigen bindingdomain of one of said KIR-CAR said TCAR, is an scFv, and the othercomprises a single VH domain, e.g., a camelid, shark, or lamprey singleVH domain, or a single VH domain derived from a human or mouse sequenceor a non-antibody scaffold, e.g., a fibronectin, e.g., a fibronectintype III antibody-like molecule, (vii) the antigen binding domain of oneof said KIR-CAR said TCAR, is an scFv, and the other comprises ananobody, or (viii) wherein, the antigen binding domain of one of saidKIR-CAR said TCAR, is an scFv, and the other comprises a camelid VHHdomain. In one embodiment, the antigen binding domain of one of saidKIR-CAR said TCAR does not comprise a light chain variable domain and aheavy chain variable domain.

In one embodiment, the antigen-binding domain of one of said KIR-CARsaid TCAR is an scFv, and the other is other than an scFv. In oneembodiment, when present on the surface of a cell, the antigen bindingdomains of said KIR-CAR said TCAR, associate with one another less thanif both were scFv antigen binding domains. In one embodiment, whenpresent on the surface of a cell, binding of the antigen binding domainof said KIR-CAR to its cognate antigen is not substantially reduced bythe presence of said second TCAR. In one embodiment, the antigen bindingdomain of one of said KIR-CAR said TCAR, comprises a single VH domain,e.g., a camelid, shark, or lamprey single VH domain, or a single VHdomain derived from a human or mouse sequence or a non-antibodyscaffold, e.g., a fibronectin, e.g., a fibronectin type IIIantibody-like molecule. In one embodiment, the antigen binding domain ofone of said KIR-CAR said TCAR, is an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence or anon-antibody scaffold, e.g., a fibronectin, e.g., a fibronectin type IIIantibody-like molecule. In one embodiment, the antigen binding domain ofone of said KIR-CAR said TCAR, is an scFv, and the other comprises ananobody. In one embodiment, the antigen binding domain of one of saidKIR-CAR said TCAR, is an scFv, and the other comprises a camelid VHHdomain.

In another aspect, the invention features a cytotoxic cell, e.g., anaturally or non-naturally occurring T cell, NK cell or cytotoxic T cellor cell of an NK cell line, e.g., NK92, comprising (a) a first KIR-CARdescribed herein. In one embodiment, said cytotoxic cell is T cell. Inone embodiment, said cytotoxic cell is an NK cell. In one embodiment,said cytotoxic cell is from an NK cell line, e.g., an NK92 cell. In oneembodiment, In one embodiment, said first KIR-CAR is an actKIR-CARdescribed herein. In one embodiment, In one embodiment, said firstKIR-CAR is an inhKIR-CAR described herein.

In an embodiment, said cytotoxic cell comprises a KIR-CAR, e.g., anactKIR-CAR, and an inhibitory molecule comprising: an inhKIR cytoplasmicdomain; a transmembrane domain, e.g., a KIR transmembrane domain; and aninhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIMdomain. In an embodiment the inhibitory molecule is a naturallyoccurring inhKIR, or a sequence sharing at least 50, 60, 70, 80, 85, 90,95, or 99% homology with, or that differs by no more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, or 20 residues from, a naturally occurring inhKIR.

In an embodiment, said cytotoxic cell comprises a KIR-CAR, e.g., anactKIR-CAR, and an inhibitory molecule comprising: a SLAM familycytoplasmic domain; a transmembrane domain, e.g., a SLAM familytransmembrane domain; and an inhibitor cytoplasmic domain, e.g., a SLAMfamily domain, e.g., an SLAM family ITIM domain. In an embodiment theinhibitory molecule is a naturally occurring SLAM family member, or asequence sharing at least 50, 60, 70, 80, 85, 90, 95, or 99% homologywith, or that differs by no more than 1, 2, 3, 4, 5,

In one embodiment, the cytotoxic cell further comprises (b) a secondKIR-CAR described herein, e.g., a second KIR-CAR that is different fromsaid first KIR-CAR. In one embodiment, one of said KIR-CAR and saidsecond KIR-CAR is an actKIR-CAR and the other is an inhKIR-CAR. In oneembodiment, said actKIR-CAR is an actKIR-CAR described herein. In oneembodiment, one of said inhKIR-CAR is an inhKIR-CAR described herein. Inone embodiment, the cytotoxic cell described herein comprises actKIR-CARdescribed herein and an inhKIR-CAR described herein.

In an embodiment the cytotoxic cell further comprises an intracellularsignaling domain, e.g., an adaptor molecule, which can produce anactivating signal, e.g., which is exogenous to said cell, which canproduce an activating signal. In one embodiment, said intracellularsignaling domain comprises an ITAM motif. In one embodiment, saidintracellular signaling domain comprises a DAP 12 polypeptide comprisingDAP 12 intracellular signaling domain. In one embodiment, said DAP 12polypeptide further comprises a transmembrane domain. In one embodiment,said DAP 12 polypeptide further comprises an extracellular domain.

In an embodiment a cytotoxic cell comprises a first and second KIR-CARdescribed herein wherein (i) the antigen binding domain of one of saidfirst KIR-CAR said second KIR-CAR does not comprise a light chainvariable domain and a heavy chain variable domain, (ii) the antigenbinding domain of one of said first KIR-CAR said second KIR-CAR is anscFv, and the other is other than an scFv, (iii) when present on thesurface of a cell, the antigen binding domains of said first KIR-CAR andsaid second KIR-CAR, associate with one another less than if both werescFv antigen binding domains, (iv) when present on the surface of acell, binding of the antigen binding domain of said first KIR-CAR to itscognate antigen is not substantially reduced by the presence of saidsecond KIR-CAR, (v) the antigen binding domain of one of said firstKIR-CAR said second KIR-CAR, comprises a single VH domain, e.g., acamelid, shark, or lamprey single VH domain, or a single VH domainderived from a human or mouse sequence or a non-antibody scaffold, e.g.,a fibronectin, e.g., a fibronectin type III antibody-like molecule, (vi)the antigen binding domain of one of said first KIR-CAR said secondKIR-CAR, comprises an scFv, and the other comprises a single VH domain,e.g., a camelid, shark, or lamprey single VH domain, or a single VHdomain derived from a human or mouse sequence or a non-antibodyscaffold, e.g., a fibronectin, e.g., a fibronectin type IIIantibody-like molecule, (vii) wherein, the antigen binding domain of oneof said first KIR-CAR said second KIR-CAR, comprises an scFv, and theother comprises a nanobody, or (viii) the antigen binding domain of oneof said first KIR-CAR said second KIR-CAR, comprises an scFv, and theother comprises a camelid VHH domain.

In one embodiment, the antigen binding domain of one of said firstKIR-CAR said second KIR-CAR does not comprise a light chain variabledomain and a heavy chain variable domain. In one embodiment, theantigen-binding domain of one of said first KIR-CAR said second KIR-CARis an scFv, and the other is other than an scFv. In one embodiment, whenpresent on the surface of a cell, the antigen binding domains of saidfirst KIR-CAR said second KIR-CAR, associate with one another less thanif both were scFv antigen binding domains. In one embodiment, whenpresent on the surface of a cell, binding of the antigen binding domainof said first KIR-CAR to its cognate antigen is not substantiallyreduced by the presence of said second KIR-CAR. In one embodiment, theantigen binding domain of one of said first KIR-CAR said second KIR-CAR,comprises a single VH domain, e.g., a camelid, shark, or lamprey singleVH domain, or a single VH domain derived from a human or mouse sequenceor a non-antibody scaffold, e.g., a fibronectin, e.g., a fibronectintype III antibody-like molecule. In one embodiment, the antigen bindingdomain of one of said first KIR-CAR said second KIR-CAR, comprises anscFv, and the other comprises a single VH domain, e.g., a camelid,shark, or lamprey single VH domain, or a single VH domain derived from ahuman or mouse sequence or a non-antibody scaffold, e.g., a fibronectin,e.g., a fibronectin type III antibody-like molecule. In one embodiment,the antigen-binding domain of one of said first KIR-CAR said secondKIR-CAR, comprises an scFv, and the other comprises a nanobody. In oneembodiment, the antigen-binding domain of one of said first KIR-CAR saidsecond KIR-CAR, comprises an scFv, and the other comprises a camelid VHHdomain.

In an embodiment a cytotoxic cell comprises KIR-CARs as described hereinand further comprises a TCAR. In one embodiment, said TCAR comprises anantigen binding domain and a primary stimulation domain. In oneembodiment, said TCAR comprises a costimulation domain.

In an embodiment the cytotoxic cell, e.g., a naturally or non-naturallyoccurring T cell, NK cell or cytotoxic T cell or NK cell line, e.g., anNK92 cell, comprises a nucleic acid as described herein; or a KIR-CARencoded by a nucleic acid described herein. In one embodiment, saidcytotoxic cell is T cell. In one embodiment, said cytotoxic cell is anNK cell. In one embodiment, said cytotoxic cell is from an NK cell line,e.g., NK92.

In another aspect, the invention features methods of making a celldescribed herein comprising, introducing into a cytotoxic cell, anucleic acid described herein into said cell. In one embodiment, saidmethod comprises forming in a cytotoxic cell, a KIR-CAR describedherein.

In another aspect, the invention features methods of treating a subject,e.g., a method of providing an anti-tumor immunity in a mammal,comprising administering to the mammal an effective amount of a celldescribed herein. In one embodiment, said cell is autologous. In oneembodiment, said cell is allogenic. In one embodiment, the cell is Tcell, e.g., an autologous T cell. In one embodiment, the cell is anallogeneic T cell. In one embodiment, the cell is an NK cell, e.g., anautologous NK cell. In one embodiment, the cell is an allogeneic NKcell. In one embodiment, the cell is cell from an NK cell line, e.g.,NK92. In one embodiment, said mammal is a human. In one embodiment, themethod further comprises evaluating said mammal, e.g., human, for a sideeffect of said treatment. In one embodiment, said side effect comprisesacute respiratory distress syndrome, febrile neutropenia, hypotension,encephalopathy, hepatic transaminitis, seizure, or macrophage activationsyndrome. In one embodiment, the method further comprises treating saidhuman having a side effect with anti-cytokine agent, e.g., a tumornecrosis factor antagonist, e.g., a TNF-Ig fusion, e.g., etanercept, anIL-6 antagonist, e.g., an IL-6 receptor antagonist, e.g., an anti-IL6receptor antibody, e.g., tocilizumab, or a corticosteroid. In oneembodiment, treating comprises administering an anti-IL6 receptorantibody to said human. In one embodiment, the method comprises treatinga mammal, e.g., a human, having a disease associated with expression ofmesothelin or CD19. In one embodiment, the method comprises treating amammal, e.g., a human, having a disorder associated with unwanted cellproliferation, e.g., cancer. In one embodiment, said disorder ispancreatic carcinoma, mesothelioma, lung carcinoma, ovarian carcinoma,leukemia or lymphoma.

In another aspect, the invention features a purified, or non-naturallyoccurring, NCR-CAR, e.g., an activating NCR-CAR, comprising anextra-cellular antigen binding domain, a transmembrane domain, e.g., atransmembrane domain comprising a positively charged moiety, e.g., anamino acid residue comprising a positively charged moiety, e.g., apositively charged side chain or an NCR transmembrane domain, and acytoplasmic domain, e.g., a NCR cytoplasmic domain. In one embodiment,said NCR-CAR comprises an a transmembrane domain comprising a positivelycharged moiety, e.g., an amino acid residue comprising a positivelycharged moiety, e.g., a positively charged side chain, e.g., NCRtransmembrane domain, e.g., a NKp30, NKp44, or NKp46 cytoplasmic domain.In one embodiment, said NCR-CAR comprises a cytoplasmic domain which caninteract with an adaptor molecule or intracellular signaling moleculecomprising, e.g., a DAP12, FcRγ or CD3 ζ cytoplasmic domain. In oneembodiment, said NCR-CAR, e.g., a NKp30-CAR, comprises a transmembranedomain which can interact with an adaptor molecule or intracellularsignaling molecule, e.g., DAP12. In one embodiment, said NCR-CARcomprises a NKp46-CAR. In one embodiment, said NKp46-CAR, comprises atransmembrane domain comprising a positively charged moiety, e.g., anamino acid residue comprising a positively charged moiety, e.g., apositively charged side chain or, e.g., an NCR transmembrane domain,which can interact with an adaptor molecule or intracellular signalingmolecule, e.g., one having a FcRγ or CD3 ζ cytoplasmic domain. In oneembodiment, said NCR-CAR described herein further comprises a hingedomain disposed between said transmembrane domain and said anextra-cellular antigen binding domain.

In another aspect, the invention features a nucleic acid, e.g., apurified or non-naturally occurring, nucleic acid, e.g., a nucleic acidcomprising a DNA, or RNA, sequence, e.g., a mRNA, comprising a sequencethat encodes a NCR-CAR described herein. In one embodiment, the nucleicacid comprises a sequence that encodes a NKp30-CAR and optionally, anadaptor molecule or intracellular signaling molecule, e.g., DAP12. Inone embodiment, said NCR-CAR, e.g., NKp46-CAR, comprises a transmembranedomain comprising a positively charged moiety, e.g., an amino acidresidue comprising a positively charged moiety, e.g., a positivelycharged side chain or an NCR transmembrane domain which can interactwith an adaptor molecule or intracellular signaling molecule, e.g., aFcRγ or CD3 ζ molecule. In one embodiment, the nucleic acid furthercomprises sequence encoding an adaptor molecule or intracellularsignaling molecule, which e.g., comprises a DAP12, FcRγ or CD3.

In another aspect, the invention features a cytotoxic cell, e.g., anaturally or non-naturally occurring T cell, NK cell or cytotoxic T cellor NK cell line comprising a NCR-CAR described herein. In oneembodiment, the cytotoxic cell further comprises an adaptor molecule orintracellular signaling molecule, which e.g., comprises a DAP12, FcRγ orCD3 ζ cytoplasmic domain. In one embodiment, the cytotoxic cellcomprises a NKp30-CAR and optionally, an adaptor molecule orintracellular signaling molecule, e.g., DAP12. In one embodiment, saidNKp46-CAR comprises a transmembrane domain which can interact with anadaptor molecule or intracellular signaling molecule, e.g., a FcRγ orCD3 ζ molecule.

In another aspect, the invention features a method of making a celldescribed herein comprising, introducing into a cytotoxic cell, anucleic acid comprising a sequence that encodes a NCR-CAR describedherein. In one embodiment, the method comprises forming in a cytotoxiccell, a NCR-CAR described herein.

In another aspect, the invention features a method of treating asubject, e.g., a method of providing an anti-tumor immunity in a mammal,comprising administering to the mammal an effective amount of a celldescribed herein, e.g., a cell of claim described herein comprisingNCR-CAR described herein.

In another aspect, the invention features a purified, or non-naturallyoccurring, SLAMF-CAR, e.g., an inhibitory SLAMF-CAR, comprising anextra-cellular antigen binding domain, a transmembrane domain, e.g., atransmembrane domain comprising a positively charged moiety, e.g., anamino acid residue comprising a positively charged moiety, e.g., apositively charged side chain, e.g., a SLAMF transmembrane domain, and aSLAMF cytoplasmic domain. In one embodiment, said SLAMF-CAR comprises aSLAMF, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, or CD2F-10cytoplasmic domain. In one embodiment, said SLAMF-CAR further comprisesa hinge domain, disposed between said transmembrane domain and said anextra-cellular antigen binding domain.

In another aspect, the invention features a nucleic acid, e.g., apurified or non-naturally occurring, nucleic acid, e.g., a nucleic acidcomprising a DNA, or RNA, sequence, e.g., a mRNA, comprising a sequencethat encodes a SLAMF-CAR described herein.

In another aspect, the invention features a cytotoxic cell, e.g., anaturally or non-naturally occurring T cell, NK cell or cytotoxic T cellor NK cell line comprising a SLAMF-CAR described herein.

In another aspect, the invention features a method of making a cytotoxiccell comprising a SLAMF-CAR described herein, comprising, introducinginto a cytotoxic cell a nucleic acid comprising a sequence that encodesa SLAMF-CAR described herein. In one embodiment, the method comprisesforming in a cytotoxic cell, a SLAMF-CAR described herein.

In another aspect, the invention features a method of treating asubject, e.g., a method of providing an anti-tumor immunity in a mammal,comprising administering to the mammal an effective amount of a cellcomprising a SLAMF-CAR described herein.

In another aspect, the invention features a purified, or non-naturallyoccurring, FcR-CAR, e.g., CD16-CAR, e.g., an activating CD16-CAR or aCD64-CAR, e.g., an activating CD64-CAR, comprising an extra-cellularantigen binding domain, a transmembrane domain, and a CD16 or CD64cytoplasmic domain. In one embodiment, said FcR-CAR is a CD16-CAR. Inone embodiment, said FcR-CAR is a CD64-CAR. In one embodiment, saidFcR-CAR can interact with an adaptor molecule or intracellular signalingmolecule, e.g., a FcRγ or CD3 ζ domain, e.g., via a transmembranedomain, e.g., a transmembrane domain comprising a positively chargedmoiety, e.g., an amino acid residue comprising a positively chargedmoiety, e.g., a positively charged side chain or e.g., a CD16 or CD64transmembrane domain. In one embodiment, said FcR-CAR further comprisesa hinge domain, disposed between said transmembrane domain and said anextra-cellular antigen binding domain.

In another aspect, the invention features a purified or non-naturallyoccurring, nucleic acid, e.g., a nucleic acid comprising a DNA, or RNA,sequence, e.g., a mRNA comprising a sequence that encodes a FcR-CARdescribed herein. In one embodiment, the nucleic acid further comprisesan adaptor molecule or intracellular signaling molecule comprising acytoplasmic activation domain, e.g., FcRγ or CD3 ζ cytoplasmic domain.In one embodiment, said FcR-CAR and said cytoplasmic activation domainare disposed on separated nucleic acid molecules, e.g., separatevectors, e.g., separate viral vectors, e.g., separate lenti-viralvectors.

In another aspect, the invention features a cytotoxic cell, e.g., anaturally or non-naturally occurring T cell, NK cell or cytotoxic T cellor NK cell line comprising a FcR-CAR described herein. In oneembodiment, the cytotoxic cell further comprises a cytoplasmicactivation domain, e.g., FcRγ or CD3 ζ cytoplasmic domain.

In another aspect, the invention features a method of making a cellcomprising a FcR-CAR described herein, comprising, introducing into acytotoxic cell, a nucleic acid comprising a sequence that encodes aFcR-CAR described herein. In one embodiment, the method comprisesforming in a cytotoxic cell, a FcR-CAR described herein.

In another aspect, the invention features a method of treating asubject, e.g., a method of providing an anti-tumor immunity in a mammal,comprising administering to the mammal an effective amount of a cellcomprising a FcR-CAR described herein.

In another aspect, the invention features purified, or non-naturallyoccurring, Ly49-CAR comprising an extra-cellular antigen binding domain,and a transmembrane domain, e.g., a Ly49-transmembrane domain, or acytoplasmic domain, e.g., an ITIM-containing cytoplasmic domain, e.g., aLy49-cytoplasmic domain. In one embodiment, the Ly49-CAR comprises atransmembrane domain and a Ly49-cytoplasmic domain. In one embodiment,said Ly49-CAR is an activating Ly49-CAR, e.g., Ly49D or Ly49H. In oneembodiment, said Ly49-CAR comprises a positively charged transmembranedomain, e.g., a positively charged Ly49 transmembrane domain. In oneembodiment, said Ly49-CAR can interact with an ITAM-containingcytoplasmic domain, e.g., DAP 12. In one embodiment, said Ly49-CARcomprises a Ly49-transmembrane domain. In one embodiment, said KIR-CARis an inhibitory Ly49-CAR, e.g., Ly49A or Ly49C. In one embodiment, saidLy49-CAR comprises an ITIM-containing cytoplasmic domain, e.g., aLy49-cytoplasmic domain. In one embodiment, said Ly49-CAR comprises aLy49-transmembrane domain or a Ly49-cytoplasmic domain selected,independently from Ly49A-Ly49W. In one embodiment, said Ly49-CAR furthercomprises a hinge domain, disposed between said transmembrane domain andsaid an extra-cellular antigen binding domain.

In another aspect, the invention features a nucleic acid, e.g., apurified or non-naturally occurring, nucleic acid, e.g., a nucleic acidcomprising a DNA, or RNA, sequence, e.g., a mRNA, comprising a sequencethat encodes a Ly49-CAR described herein. In one embodiment, the nucleicacid further comprises a cytoplasmic activation domain, e.g., DAP12cytoplasmic domain. In one embodiment, said Ly49-CAR and saidcytoplasmic activation domain are disposed on separate nucleic acidmolecules, e.g., separate vectors, e.g., separate viral vectors, e.g.,separate lenti-viral vectors.

In another aspect, the invention features a cytotoxic cell, e.g., anaturally or non-naturally occurring T cell, NK cell or cytotoxic T cellor NK cell line comprising a Ly49-CAR described herein. In oneembodiment, the cytotoxic cell further comprises a cytoplasmicactivation domain, e.g., DAP12 cytoplasmic domain.

In another aspect, the invention features a method of making a cellcomprising a Ly49-CAR described herein, comprising, introducing into acytotoxic cell, a nucleic acid comprising a sequence that encodes aLy49-CAR described herein into said cell.

In another aspect, the invention features a method of making a cellcomprising a Ly49-CAR described herein, comprising, forming in acytotoxic cell, a Ly49-CAR described herein.

In another aspect, the invention features a method of treating asubject, e.g., a method of providing an anti-tumor immunity in a mammal,comprising administering to the mammal an effective amount of a celldescribed herein, e.g., a cell comprising a Ly49-CAR described herein.

In another aspect, the invention features a cell comprising, e.g., acytotoxic cell, comprising a first non-naturally occurring chimericmembrane embedded receptor comprising an antigen binding domain and asecond non-naturally occurring chimeric membrane embedded receptorcomprising an antigen binding domain wherein, (i) the antigen bindingdomain of one of said first and said second non-naturally occurringchimeric membrane embedded receptor does not comprise a light chainvariable domain and a heavy chain variable domain, (ii) the antigenbinding domain of one of said first and said second non-naturallyoccurring chimeric membrane embedded receptor comprises an scFv, and theother is other than an scFv, (iii) when present on the surface of acell, the antigen binding domains of said first and said secondnon-naturally occurring chimeric membrane embedded receptor, associatewith one another less than if both were scFv antigen binding domains,(iv) when present on the surface of a cell, binding of the antigenbinding domain of said first non-naturally occurring chimeric membraneembedded receptor to its cognate antigen is not substantially reduced bythe presence of said second non-naturally occurring chimeric membraneembedded receptor, (v) the antigen binding domain of one of said firstand said second non-naturally occurring chimeric membrane embeddedreceptor, comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence or a non-antibody scaffold, e.g., a fibronectin, e.g., afibronectin type III antibody-like molecule, (vi) the antigen bindingdomain of one of said first said second non-naturally occurring chimericmembrane embedded receptor, comprises an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence or anon-antibody scaffold, e.g., a fibronectin, e.g., a fibronectin type IIIantibody-like molecule, (vii) the antigen binding domain of one of saidfirst and said second non-naturally occurring chimeric membrane embeddedreceptor, comprises an scFv, and the other comprises a nanobody, and(viii) the antigen binding domain of one of said first and said secondnon-naturally occurring chimeric membrane embedded receptor, comprisesan scFv, and the other comprises a camelid VHH domain. In oneembodiment, said cell is T cell. In one embodiment, said cell is an NKcell. In one embodiment, said cell is from an NK cell line, e.g., NK92.In one embodiment, one of said first and said second non-naturallyoccurring chimeric membrane embedded receptors is a TCAR. In oneembodiment, both of said first and said second non-naturally occurringchimeric membrane embedded receptors is a TCAR. In one embodiment, oneof said first and said second non-naturally occurring chimeric membraneembedded receptors is a NKR-CAR, e.g., a KIR-CAR. In one embodiment,both of said first and said second non-naturally occurring chimericmembrane embedded receptors is a NKR-CAR, e.g., a KIR-CAR. In oneembodiment, the antigen binding domain of one of said first and saidsecond non-naturally occurring chimeric membrane embedded receptor doesnot comprise a light chain variable domain and a heavy chain variabledomain. In one embodiment, when present on the surface of a cell, theantigen binding domains of said first and said second non-naturallyoccurring chimeric membrane embedded receptor, associate with oneanother less than if both were scFv antigen binding domains. In oneembodiment, when present on the surface of a cell, binding of theantigen binding domain of said first non-naturally occurring chimericmembrane embedded receptor to its cognate antigen is not substantiallyreduced by the presence of said second non-naturally occurring chimericmembrane embedded receptor. In one embodiment, the antigen bindingdomain of one of said first and said second non-naturally occurringchimeric membrane embedded receptor, comprises a single VH domain, e.g.,a camelid, shark, or lamprey single VH domain, or a single VH domainderived from a human or mouse sequence or a non-antibody scaffold, e.g.,a fibronectin, e.g., a fibronectin type III antibody-like molecule. Inone embodiment, the antigen binding domain of one of said first saidsecond non-naturally occurring chimeric membrane embedded receptor,comprises an scFv, and the other comprises a single VH domain, e.g., acamelid, shark, or lamprey single VH domain, or a single VH domainderived from a human or mouse sequence or a non-antibody scaffold, e.g.,a fibronectin, e.g., a fibronectin type III antibody-like molecule. Inone embodiment, the antigen binding domain of one of said first and saidsecond non-naturally occurring chimeric membrane embedded receptor,comprises an scFv, and the other comprises a nanobody. In oneembodiment, the antigen binding domain of one of said first and saidsecond non-naturally occurring chimeric membrane embedded receptor,comprises an scFv, and the other comprises a camelid VHH domain. In oneembodiment, the invention comprises a nucleic acid, e.g., a purified ornon-naturally occurring, nucleic acid, comprising a sequence thatencodes a first and second non-naturally occurring chimeric membraneembedded receptor comprising an antigen binding domain described herein.In one embodiment, the invention comprises a method of making a celldescribed herein comprised introducing into a cell the nucleic aciddescribed herein. In one embodiment, the invention comprises a method ofmaking a cell described herein, comprising, forming in a cytotoxic cell,a first and said second non-naturally occurring chimeric membraneembedded receptor described herein. In one embodiment, the inventioncomprises a method of treating a subject e.g., a method of providing ananti-tumor immunity in a mammal, comprising administering to the mammalan effective amount of a cell described herein.

In another aspect, the invention features a kit comprising a cell ornucleic acid described herein.

In another aspect, the invention features an isolated nucleic acidsequence encoding a KIR-CAR (killer cell immunoglobulinreceptor-like-chimeric antigen receptor), wherein the isolated nucleicacid sequence comprises the nucleic acid sequence of an antigen bindingdomain and a KIR or fragment thereof. In one embodiment, the antigenbinding domain is selected from the group consisting of a murineantibody, a humanized antibody, a human antibody, a chimeric antibody,and a fragment thereof. In one embodiment, the fragment is a Fab or anscFv. In one embodiment, the KIR is selected from the group consistingof an activating KIR, an inhibitory KIR, and any combination thereof. Inone embodiment, at least one hinge region has been removed from theactivating KIR.

In another aspect, the invention features an isolated KIR-CAR (killercell immunoglobulin-like receptor-chimeric antigen receptor) comprisingan antigen binding domain and a KIR or fragment thereof. In oneembodiment, the antigen binding domain is selected from the groupconsisting of a murine antibody, a humanized antibody, a human antibody,a chimeric antibody, and a fragment thereof. In one embodiment, thefragment is a Fab or an scFv. In one embodiment, the KIR is selectedfrom the group consisting of an activating KIR, an inhibitory KIR, andany combination thereof. In one embodiment, at least one hinge regionhas been removed from the activating KIR. In another aspect, theinvention features a composition comprising at least two KIR-CARs,wherein the first KIR-CAR comprises an antigen binding domain and anactivating KIR or fragment thereof and the second KIR-CAR comprises anantigen binding domain and an inhibitory KIR or fragment thereof. In oneembodiment, the antigen binding domain in the first KIR-CAR is specificfor an antigen present on a tumor and the antigen binding domain in thesecond KIR-CAR is specific for an antigen present on a normal cell.

In another aspect, the invention features a genetically modified T cellcomprising at least two KIR-CARs, wherein the first KIR-CAR comprises anantigen binding domain and an activating KIR or fragment thereof and thesecond KIR-CAR comprises an antigen binding domain and an inhibitory KIRor fragment thereof. In one embodiment, the antigen binding domain inthe first KIR-CAR is specific for an antigen present on a tumor and theantigen binding domain in the second KIR-CAR is specific for an antigenpresent on a normal cell. In one embodiment, the cell is a T cell.

In another aspect, the invention features a method of providing ananti-tumor immunity in a mammal, the method comprising administering tothe mammal an effective amount of a cell comprising at least twoKIR-CARs, wherein the first KIR-CAR comprises an antigen binding domainand an activating KIR or fragment thereof and the second KIR-CARcomprises an antigen binding domain and an inhibitory KIR or fragmentthereof. In one embodiment, the antigen binding domain in the firstKIR-CAR is specific for an antigen present on a tumor and the antigenbinding domain in the second KIR-CAR is specific for an antigen presenton a normal cell, thereby controlling the off-target activity of thecell. In one embodiment, the cell is a T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A and 1B are a series of schematics showing the structure ofnaturally occurring inhibitory and activating KIRs (FIG. 1A) and ascFv-based activating KIR-CAR (FIG. 1B).

FIG. 2 is a schematic representation of the lentiviral vector used todeliver an activating KIR-based CAR in combination with the DAP12signaling molecule.

FIG. 3 is an image demonstrating that a mesothelin-specific actKIR-CARscan be efficiently expressed on the surface of primary human T cells.Human T cells were stimulated with anti-CD3/anti-CD28 microbeads andtransduced with the indicated CAR or mock transduced and expanded exvivo. The expression was detected using a biotinylated goat-anti-mouseF(ab)2-specific polyclonal IgG (Jackson Immunologics) followed bystaining with streptavidin-PE.

FIG. 4 is an image demonstrating that T cells expressing the SS 1actKIR-CAR exhibited cytotoxic activity towards target K562 cellsengineered to express the mesothelin ligand (KT-meso). Human T cellswere stimulated with anti-CD3/anti-CD28 microbeads, transduced with theindicated CAR or mock transduced and expanded ex vivo. 10⁵ CFSE-labeledK562 cells expressing mesothelin (KT-meso) or wild-type control K562were incubated with varying ratios of CAR-expressing T cells for 16hours at 37° C., 5% CO₂. The K562 target cells were then enumerated byflow cytometry using countbright beads and a viability stain (7AAD). Thepercentage of K562 cells lysed (percent lysis) was calculated bysubtracting the number of viable target cells remaining after incubationwith effector T cells from the number of viable K562 remaining afterovernight culture without effector T cells, and then dividing by thenumber of viable K562 remaining after overnight culture without effectorT cells.

FIGS. 5A and 5B are a series of schematics showing an activating KIR CARin which the KIR2DS2 hinge was removed (KIR2S CAR). Based upon thekinetic segregation model of TCR activation diagrammed in FIG. 5A, it isbelieved that the mesothelin-specific SS 1 KIR CAR has a hinge that istoo long to permit appropriate segregation. Therefore making themesothelin-specific KIR CAR hinge shorter is believed to improve thefunction. FIG. 5B is a schematic showing that the SS 1 scFv was fused tothe KIR transmembrane domain without the two Ig-like domains fromKIR2DS2 as the hinge.

FIGS. 6A and 6B are a series of images demonstrating that SS1 scFv basedKIRS2 CAR exhibits enhanced cytolytic activity towardsmesothelin-expressing target cells compared with the CAR formed byfusion of the SS1 scFv onto full length wildtype KIR2DS2. Primary humanT cells were stimulated with CD3/28 microbeads followed by lentiviraltransduction with either the SS1-KIR2DS2 activating KIR-CAR, SS1-KIRS2activating KIR CAR, the SS1-zeta CAR. Mock non-transduced T cells (NTD)were used as a control. The T cells were expanded until the end oflog-phase growth. The surface expression of the SS1-specific CARs wasdetermined by flow cytometry using a biotinylated goat anti-mouse F(ab)2specific polyclonal antibody followed by streptavidin-PE detection asshown in FIG. 6A. Shown in FIG. 6B, K562 target cells with or withoutmesothelin and stained with CFSE were mixed with the effector T cellscharacterized in FIG. 6A as indicated using varying effector T cell totarget ratios ranging from 10:1 to 1:1. Target K562 cell lysis wasassessed using flow cytometry to determine the % of viable CFSE+ cellsas described for FIG. 4. Data shown is the calculated % target celllysis compared against target cells without effector cells.

FIGS. 7A and 7B are a series of images showing co-expression of the CD19actKIR-CAR and the SS 1 inhKIR-CAR. Jurkat NFAT-GFP reporter cells weretransduced with the indicated KIR CAR or non-transduced (NDT) and mixed1:1 with target cells with or without the CD19 and mesothelin antigensas indicated. Results shows GFP expression at 24 hours following mixingof Jurkat and Target cells (FIG. 7A). FIG. 7B shows surface expressionof the mesothelin and CD19 idiotypes as determined by staining with amesothelin-Fc fusion protein and a monoclonal antibody specific for theFMC63 anti-CD19 scFv idiotype.

FIGS. 8A through 8C are a series of images demonstrating co-expressionof wild-type PD-1 with both an activating KIR-based CAR or TCR-zetabased CAR targeting mesothelin. Primary human T cells were stimulatedwith CD3/28 microbeads followed by lentiviral transduction with eitherthe SS1-KIRS2 activating KIR CAR or the SS1-zeta CAR. Mocknon-transduced cells (NTD) were used as a negative control. The T cellswere expanded over 9 days, and surface CAR expression was determined bystaining with mesothelin-Fc followed by a goat-anti-human Fc specificantibody conjugated to PE (FIG. 8A). K562 cell lines (wildtype [wt],mesothelin expressing [meso] or mesothelin and PD-L1 co-expressing[meso-PDL1]) were stained using the CAK1 anti-mesothelin specificmonoclonal antibody to confirm mesothelin expression on the targets(FIG. 8B). The primary human T cells transduced as shown in FIG. 8A wereelectroporated with 10 ug of in vitro transcribed RNA encoding wild-typePD1 using a BTX ECM830 electroporator (PD1+) or mock transfected (PD1−).The surface expression of PD-1 was expressed using an anti-PD 1monoclonal conjugated to APC (FIG. 8C).

FIG. 9 is an image demonstrating that the combination of co-expressingwild-type PD-1 with both an activating KIR-based CAR and TCR-zeta basedCAR targeting mesothelin led to PD-1 ligand 1 (PDL-1) dependentinhibition of the mesothelin-specific activating KIR-CAR cytotoxicity.Primary human T cells were stimulated with CD3/28 microbeads followed bylentiviral transduction with either the SS1-KIRS2 activating KIR CAR,the SS1-zeta CAR or mock transduced (NTD). The T cells were expandedover 9 days followed by electroporation of 5×10⁶ T cells with 10 ug ofin vitro transcribed RNA encoding wild-type PD1 using a BTX ECM830electroporator (PD1+) or mock transfected (PD1−). The surface expressionof the SS1-specific CAR and PD-1 was determined as shown in FIG. 8. K562target cells with either no mesothelin or expressing mesothelin with orwithout PDL-1 were mixed with the different T cells conditions asindicated using varying effector T cell to target ratios of 30:1 to 1:1as shown. Target K562 cell lysis was assessed using a calcein AM dyemethod to quantify the remaining viable cells following 4 hours ofincubation. Data shown is calculated % target cell lysis comparedagainst target cells without effector cells.

FIG. 10 is an image demonstrating the interferon-gamma (IFN-γ)production by T cells from two different donors expressing amesothelin-specific activating KIR-based CAR (SS1-KIR2DS2 or SS1-KIRS2)or TCR-zeta based CAR with or without a costimulatory domain (SS1-z,SS1-28z or SS1-BBz). Primary human T cells were stimulated with CD3/28microbeads followed by lentiviral transduction with the indicatedactivating KIR CAR or TCR-zeta based CAR. Mock non-transduced cells(NTD) were used as a negative control. K562 target cells with either nomesothelin (K562) or expressing mesothelin (K562-meso) were mixed withthe different T cells conditions as indicated at a 2:1 ratio of effectorT cells to target cells. Following 16 hours of incubation, IFN-γ wasmeasured in the culture supernatants using a human IFN-gamma specificELISA assay (R&D systems).

FIG. 11 is an image demonstrating the interleukin-2 (IL-2) production byT cells expressing a mesothelin-specific activating KIR-based CAR(SS1-KIR2DS2 or SS1-KIRS2) or TCR-zeta based CAR with or without acostimulatory domain (SS1-z, SS1-28z or SS1-BBz). Primary human T cellswere stimulated with CD3/28 microbeads followed by lentiviraltransduction with the indicated activating KIR CAR or TCR-zeta basedCAR. Mock non-transduced cells (NTD) were used as a negative control.K562 target cells with either no mesothelin (K562) or expressingmesothelin (K562-meso) were mixed with the different T cells conditionsas indicated at a 2:1 ratio of effector T cells to target cells.Following 16 hours of incubation, IL-2 was measured in the culturesupernatants using a human IL-2 specific ELISA assay (R&D systems).

FIGS. 12A-12B depict construction of a mesothelin-specific KIR-basedchimeric antigen receptor (KIR-CAR) engineered T cell with robustcytotoxic activity. Primary human T cells were stimulated with CD3/28microbeads followed by transduction with a lentiviral vector expressingeither GFP and dsRed (Control) or DAP12 and dsRed (DAP12). The cellswere expanded ex vivo until the end of log phase growth. 5×10⁶ T cellsfrom each transduced population were electroporated with 10 ug of invitro transcribed RNA encoding SS1-KIRS2 using a BTX ECM830electroporator. The expression of both dsRed and SS1-KIRS2 was assessedby flow cytometry with the SS1-KIRS2 detected using a biotinylated goatanti-mouse F(ab)2 specific polyclonal antibody followed bystreptavidin-PE. The upper panel of FIG. 12A shows the gating strategyfor identification of T cells expressing dsRed, which were then analyzedfor SS1-KIRS2 expression as shown in the lower portion of the panel.FIG. 12B shows the ability of the cells characterized in FIG. 12A tomediate cytotoxicity against wild-type K562 cells (K562-wt) or K562cells that express mesothelin (K562-mesothelin) as assessed using a 4-hr⁵¹Cr-release assay.

FIG. 13 shows that the expression of an endogenous TCR is unaffected bySS1-KIRS2 and DAP12 expression. 5×10⁶ primary human T cells wereelectroporated with 10 ug of in vitro transcribed RNA encoding SS1-KIRS2or mock transfected using a BTX ECM830 electroporator. After overnightincubation, the transfected T cells were stained for the expression ofSS1-KIRS2 using a biotinylated goat anti-mouse F(ab)2 specificpolyclonal antibody followed by streptavidin-PE. The expression ofVβ13.1 was assessed using a PE-conjugated monoclonal antibody specificto this Vβ chain of the TCR.

FIG. 14 illustrates the ability of a mesothelin-specific KIR-based CAR(SS1-KIRS2) to stimulate T cell proliferation that is antigen-dependentbut independent of additional CD28 costimulation. Primary human T cellswere stimulated with CD3/28 microbeads followed by lentiviraltransduction of SS1-KIRS2 and DAP12 or the mesothelin-specific TCR-zetaCAR (SS1-zeta). Mock non-transduced cells (NTD) were used as a negativecontrol. K562 target cells with either no mesothelin (K562 wt) orexpressing mesothelin (K562-mesothelin) were mixed with the different Tcells conditions as indicated at a 2:1 ratio of effector T cells totarget cells. T cells stimulated with K562-mesothelin were furtherdivided into a condition with or without a monoclonal anti-CD28 agonistantibody (clone 9.3) at 1 ug/mL. The number of viable T cells wereenumerated by flow cytometry using bead-based counting at the indicatedtime points to calculate the number of population doublings followingantigen stimulation.

FIGS. 15A-15C demonstrate that mesothelin-specific KIR-CAR modified Tcells show enhanced anti-tumor activity in vivo compared with secondgeneration TCR-ζ based CARs bearing CD28 or CD137 (4-1BB) costimulatorydomains. FIG. 15A shows an experiment in which NOD-SCID-γ_(c) ^(−/−)(NSG) mice were subcutaneously implanted with a mesothelioma-derivedcell expressing mesothelin (EM-meso cells). 20 days following tumorimplantation, each animal was injected intravenously with 7 million Tcells that were stimulated with anti-CD3/anti-CD28 stimulator beadsfollowed lentiviral transduction with a series of CD3ζ-based CAR with orwithout a costimulatory domain (SS1-ζ, SS1-BBζ and SS1-28ζ) or themesothelin-specific KIR-based CARs, SS1-KIRS2 with DAP12. Mocktransduced T cells (NTD) were used as a control. Tumor volume wasassessed via caliper measurement. 8 animals were analyzed for each Tcell condition FIG. 15B shows that the in vivo activity of the KIR-CARis independent of T cell engraftment in blood, spleen or tumor. Thefrequency of human CD45+ T cells was assessed at the end of theexperiment by flow cytometry, and data are expressed as a percentage oftotal viable cells in the blood, spleen and tumor digest. FIG. 15C showsthat DAP12-modified T cells require the mesothelin-specific KIR-basedCAR for tumor eradication. The same model as that shown in FIG. 15A wasused. 4 million T cells expressing DAP12 and dsRed (DAP12), SS1-28z orSS1-KIRS2 and DAP12 (SS1-KIRS2) were injected intravenously on day 20,and tumor volume was assessed over time via caliper measurement.

FIGS. 16A-16B demonstrate a KIR-based CAR with CD19 specificity cantrigger antigen-specific target cell cytotoxicity. Followinganti-CD3/anti-CD28 bead activation, T cells were transduced with abicistronic lentiviral vector expressing DAP12 along with either aCD19-specific KIR-based CAR in which the FMC63-derived scFv is fused tofull length KIR2DS2 (CD19-KIR2DS2) or a KIR-based CAR generated byfusing the FMC63 scFv to the transmembrane and cytoplasmic domain ofKIR2DS2 via a short linker [Gly]₄-Ser linker (SEQ ID NO: 66)(CD19-KIRS2). The transduced T cells were cultured until the end of thelog phase growth, and the expression of the CD19-specific KIR-based CARwas assessed by flow cytometry using a biotinylated goat-anti-mouseF(ab)₂ polyclonal antibody followed by SA-PE. ⁵¹Cr-labeled K562 targetcells with (K562-CD19) or without (K562-wt) CD19 expression were mixedat varying ratios with T cells to target cells (E:T ratio). Cytotoxicitywas determined by measuring the fraction of ⁵¹Cr released into thesupernatant at 4 hours. Control T cells that were either mock transduced(NTD) or transduced with a CD3ζ-based CAR specific to CD19 (CD19-z) werealso included as negative and positive controls, respectively.

FIGS. 17A-17B show CD19-KIRS2 in vivo activity. NOD-SCID-γ_(c) ^(−/−)(NSG) mice were engrafted intravenously by tail vein injection on day 0of 1 million Nalm-6 CBG tumor cells, a leukemic cell line expressingCD19. T cells were stimulated with anti-CD3/anti-CD28 stimulator beadsfollowed by lentiviral transduction on day 1 with a series ofCD19-specific CD3ζ-based CAR with or without a costimulatory domain(CD19z, 19BBz) or the CD19-specific KIR-based CARs, CD19-KIRS2 withDAP12 (19KIRS2). Mock non-transduced T cells (NTD) were used as acontrol. The T cells were expanded until the end of log-phase growth exvivo and injected intravenously on day 5 post leukemic cell lineinjection with 2 million CAR T cells per mouse. Tumor burden wasassessed via bioluminescent imaging. 5 animals were analyzed for each Tcell condition. FIG. 17A shows the individual bioluminescent photon fluxfor individual animals on day 5 (baseline prior to T cell injection) andat day 15 following leukemic cell engraftment. FIG. 17B shows the mediantotal flux for each treatment group over time.

FIGS. 18A-18B demonstrate an NKp46-based NCR CAR with mesothelinspecificity triggers antigen specific cytotoxicity. Followinganti-CD3/anti-CD28 bead activation, T cells were transduced with abi-cistronic lentiviral vector expressing either DAP12 and SS1-KIRS2(control), or FcεRγ and a mesothelin specific NKp46-based CAR(SS1-NKp46) or FcεRγ and a mesothelin-specific NKp46 CAR in which thenatural NKp46 extracellular domain was truncated (SS1-TNKp46). Theexpression of the mesothelin-specific CARs was assessed by flowcytometry using a biotinylated goat-anti-mouse F(ab)2 polyclonalantibody followed by SA-PE as shown in FIG. 18A. The T cells were mixedwith ⁵¹Cr-labeled K562 target cells expressing mesothelin at varyingratios of effector T cells to target K562 cells (E:T ratio).Cytotoxicity was determined by measuring the fraction of ⁵¹Cr releasedinto the supernatant at 4 hours compared with spontaneous release asshown in FIG. 18B.

FIG. 19 shows a schematic representation of the receptors used inExperiments shown in FIGS. 21-23.

FIG. 20 demonstrates the generation and characterization of a K562-mesocell line that express the KIR2DL3 ligand HLA-Cw. K562 cells (K562) orK562 cells expressing mesothelin (K562-meso) were transduced with theHLA-Cw3 allele followed by fluorescence activated cell sorting to obtainK562 cells expressing HLA-Cw with (K562-meso-HLACw) or without(K562-HLACw) expression of mesothelin. HLA-Cw3 expression was assessedby flow cytometry using an APC-conjugated monoclonal antibody thatrecognizes HLA-A, B and C alleles (clone W6/32).

FIG. 21 demonstrates co-expression of SS1-KIRS2 and KIR2DL3 in primaryhuman T cells. Primary human T cells were stimulated with CD3/28microbeads followed by lentiviral transduction with SS1-KIRS2 and DAP12(SS1-KIRS2) or mock transduced (NTD) in combination with wild-typeKIR2DL3. The T cells were expanded until the end of log-phase growth.The surface expression of the mesothelin-specific CAR and KIR2DL3 wasdetermined by staining with mesothelin-Fc followed by PE-conjugatedgoat-anti-human Fc and a monoclonal antibody to the KIR2DL3 ectodomain.

FIG. 22 demonstrates that KIR2DL3 coexpressed with a KIR CAR cansuppress antigen specific cytotoxicity in the presence of HLA-Cw on thetarget cells. T cell that were generated and characterized as describedin FIG. 21 were mixed with 51-Cr labeled target K562 cells that weregenerated and characterized as described in FIG. 22. Cytotoxicity wasdetermined by measuring the fraction of ⁵¹Cr released into thesupernatant at 4 hours compared target cells without effector cells.

FIG. 23 shows a schematic representation of the receptors used inExperiments shown in FIG. 24.

FIG. 24 demonstrates the inability to co-express two scFv-based chimericreceptors on the surface of the T cell while retaining each receptors'respective binding specificity. Jurkat T cells were transduced using alentiviral vector encoding SS1-KIR2DL3. These cells were subsequentlytransduced with a second lentiviral vector encoding CD19-KIR2DS2 atvarying dilutions of the vector. The expression of the SS1-specific scFvwas assessed using mesothelin-Fc followed by PE-conjugatedgoat-anti-human Fc. The CD19-specific scFv expression was assessed usinga PE-conjugated monoclonal antibody specific to the FMC63 idiotype.

FIG. 25 is an image demonstrating that expression of a CD19-specific CARalso reduced the expression of mesothelin-binding sites on the surfaceof cells co-expressing an SS1-zeta-mCherry fusion CAR. Primary human Tcells were stimulated with CD3/28 microbeads followed by lentiviraltransduction with an SS 1 scFv zeta CAR bearing a C-terminal mCherryfusion (SS1z-mCh) or the FMC63-derived CD19 specific 41BB-zeta CAR(19bbz) alone or combination. Mock-transduced cells were used as acontrol. The T cells were expanded until the end of log-phase growth,and dsRed as well as surface CAR expression was determined by flowcytometry after staining with mesothelin-Fc followed by agoat-anti-human Fc specific polyclonal antibody conjugated to FITC.

FIG. 26 is an image demonstrating that mutually exclusive expression ofbinding sites for the SS 1 scFv is not unique to the FMC63 scFv. Primaryhuman T cells were stimulated with CD3/28 microbeads followed bylentiviral transduction with either an SS1 scFv zeta CAR or various CD19specific 41BB-zeta CARs (19BBz [FMC63 scFv, 214d scFv or the BL22 scFvCARs with alternate VH and VL orientations [H2L and L2H]). NTDrepresents mock-transduced cells used as a staining control. Inaddition, a separate set of T cells were co-transduced with the SS 1scFv zeta CAR and the different CD19 specific CARs as above. The T cellswere expanded until the end of log phase growth, and surface CARexpression was determined by staining with biotinylated protein L(recognizes kappa light chain) followed by streptavidin APCsimultaneously with mesothelin-Fc followed by a goat-anti-human Fcspecific polyclonal antibody conjugated to PE. The cotransduced cellsshowed that the mutually exclusive expression observed with FMC63-basedCAR is also observed with other scFv-CARs.

FIGS. 27A-27B depict the putative mechanism for loss of scFv bindingwhen two scFv molecules are co-expressed on the cell surface (FIG. 27A)and the putative avoidance of this interaction when a camelid single VHHdomain-based CAR is expressed on a T cell surface in combination with ascFv-based CAR.

FIG. 28 demonstrates a camelid single VHH domain-based CAR can beexpressed on a T cell surface in combination with a scFv-based CARwithout appreciable receptor interaction. Jurkat T cells expressing GFPunder an NFAT-dependent promoter (NF-GFP) were transduced with either amesothelin-specific activating CAR (SS1-CAR), CD19-specific activating(19-CAR) or a CAR generated using a camelid VHH domain specific to EGFR(VHH-CAR). Following transduction with the activating CAR, the cellswere then transduced with an additional inhibitory CAR recognizing CD19(19-PD1) to generate cells co-expressing both the activating andinhibitory CAR (SS1+19PD1, 19+19PD1 or VHH+19PD1). The transduced JurkatT cells were co-cultured for 24 hours with different cell lines that areeither 1) devoid of all target antigens (K562), 2) express mesothelin(K-meso), CD19 (K-19) or EGFR (A431) only, 3) express a combination ofEGFR and mesothelin (A431-mesothelin) or CD19 (A431-CD19) or 4) expressa combination of CD19 and mesothelin (K-19/meso). Additional conditionsthat include either no stimulator cells (no stim) or K562 with 1 ug/mLof OKT3 (OKT3) were also included as negative and positive controls forNFAT activation, respectively. GFP expression, as a marker of NFATactivation, was assessed by flow cytometry.

FIGS. 29A-29B show a KIR2DS2 Sequence Annotation (SEQ ID NOS 52 and 53,respectively, in order of appearance). SEQ ID NO: 52 depicts thenucleotide sequence, and SEQ ID NO: 53 depicts the amino acid sequence.

FIGS. 30A-30C show a KIR2DL3 Sequence Annotation (SEQ ID NOS 54 and 55,respectively, in order of appearance). SEQ ID NO: 54 depicts thenucleotide sequence and SEQ ID NO: 55 depicts the amino acid sequence.

FIGS. 31A-31B show a NKp46 Sequence Annotation (SEQ ID NOS 56 and 57,respectively, in order of appearance). SEQ ID NO: 56 depicts thenucleotide sequence and SEQ ID NO: 57 depicts the amino acid sequence.

FIGS. 32A-32B show a SS1-KIRS2 Sequence Annotation (SEQ ID NOS 58 and59, respectively, in order of appearance). SEQ ID NO: 58 depicts thenucleotide sequence and SEQ ID NO: 59 depicts the amino acid sequence.

FIGS. 33A-33C show a SS1-KIR2DS2 Sequence Annotation (SEQ ID NOS 60 and61, respectively, in order of appearance). SEQ ID NO: 60 depicts thenucleotide sequence and SEQ ID NO: 61 depicts the amino acid sequence.

FIGS. 34A-34B show a SS1-tNKp46 Sequence Annotation (SEQ ID NOS 62 and63, respectively, in order of appearance). SEQ ID NO: 62 depicts thenucleotide sequence and SEQ ID NO: 63 depicts the amino acid sequence.

FIGS. 35A-35B show a SS1-KIRL3 Sequence Annotation (SEQ ID NOS 64 and65, respectively, in order of appearance). SEQ ID NO: 64 depicts thenucleotide sequence and SEQ ID NO: 65 depicts the amino acid sequence.

DETAILED DESCRIPTION

In one aspect, the present invention provides compositions and methodsfor regulating the specificity and activity of T cells, or othercytotoxic cells, e.g., NK cells. In an embodiment a chimeric antigenreceptor (a CAR), e.g., a NK cell receptor CAR (a NKR-CAR) based on anNK cell receptor (a NKR), e.g., a KIR-CAR, a NCR-CAR, a SLAMF-CAR, aFcR-CAR, or a Ly49-CAR is provided. In one embodiment, the inventionprovides a type of chimeric antigen receptor (CAR) wherein the CAR istermed an NKR, e.g., a “KIR-CAR,” which is a CAR design comprising acomponent of a receptor found on natural killer (NK) cells. In oneembodiment, the NK receptor includes but is not limited to a killer cellimmunoglobulin-like receptor (KIR). KIRs can function as an activatingKIR or an inhibiting KIR.

One advantage of the NKR-CARs, e.g., KIR-CARs, of the invention is thata NKR-CAR, e.g., a KIR-CARs provides a method for regulating cytotoxiccell, e.g., T cell, specificity to control off-target activity of theengineered T cell. In some instances, the KIR-CARs of the invention donot require a costimulation to proliferate.

NKR-CARs can deliver a signal through an adaptor protein, e.g., an ITAMcontaining adaptor protein. In one embodiment, the KIR-CARs of theinvention comprise an activating KIR which delivers its signal throughan interaction with the immunotyrosine-based activation motif (ITAM)containing membrane protein, DAP12 that is mediated by residues withinthe transmembrane domains of these proteins.

In an embodiment a NKR-CAR can deliver an inhibitory signal by means ofan inhibitory motif. In one embodiment, the KIR-CARs of the inventioncomprise an inhibitory KIR which delivers its signal through aninteraction with the immunotyrosine-based inhibitory motifs (ITIMs).KIRs bearing cytoplasmic domains that contain ITIMs abrogate theactivating signal leading to inhibition of NK cytolytic and cytokineproducing activity. However, the invention should not be limited toinhibitory KIRs. Rather, any inhibitory protein having a cytoplasmicdomain that is associated with an inhibitory signal can be used in theconstruction of the CARs of the invention.

Accordingly, the invention provides a composition comprising a NKR-CAR,e.g., a KIR-CAR, vectors comprising the same, compositions comprising aNKR-CAR, e.g., a KIR-CAR, vectors packaged in viral particles, andrecombinant T cells or other cytotoxic cells comprising a NKR-CAR, e.g.,a KIR-CAR. The invention also includes methods of making a geneticallymodified T cell or other cytotoxic cell, e.g., a NK cell, or cultured NKcell, e.g., a NK92 cell, expressing a NKR-CAR, e.g., a KIR-CAR(KIR-CART), wherein the expressed NKR-CAR, e.g., a KIR-CAR, comprises anantigen recognition domain of a specific antibody with an intracellularsignaling molecule from a NKR, e.g., a KIR. For example, in someembodiments, the intracellular signaling molecule includes, but is notlimited to, a KIR ITAM, a KIR ITIM, and the like.

Accordingly, the invention provides compositions and methods to regulatethe specificity and activity of T cells or other cytotoxic cellsmodified to express a NKR-CAR, e.g., a KIR-CAR. The present inventionalso provides cells comprising a plurality of types of NKR-CARs, e.g.,KIR-CARs (e.g. activating NKR-CARs, e.g., KIR-CARs and inhibitingNKR-CAR, e.g., a KIR-CAR), wherein the plurality of types of NKR-CARs,e.g., KIR-CARs, participate in signaling to regulate T cell activation.In this aspect, it is beneficial to effectively control and regulateNKR-CAR cytotoxic cells, e.g., KIR-CAR T cells, such that they killtumor cells while not affecting normal bystander cells. Thus, in oneembodiment, the present invention also provides methods of killingcancerous cells while minimizing the depletion of normal non-cancerouscells, thereby improving the specificity of a NKR-CAR, e.g., a KIR-CAR,therapy.

In one embodiment, the NKR-CAR, e.g., KIR-CAR approach includes thephysical separation of a plurality of types of CARs expressed on a cell,wherein binding of a plurality of types of NKR-CARs, e.g., KIR-CARs totheir target antigen is required for NKR-CAR cytotoxic cell, e.g.,KIR-CAR T cell, activation. For example in the KIR-CAR approach, eachKIR-CAR from the plurality of type of KIR-CARs have differentintracellular signaling domain. For example, when a plurality of typesof KIR-CARs is used to induce KIR-CAR T cell activation, the first typeof KIR-CARs can only comprise an intracellular domain from an activatingKIR and the second type of CAR can only comprise an intracellular domainfrom an inhibiting KIR. In this manner, conditional activation of Tcells is generated by engagement of the activating KIR-CAR (actKIR-CAR)to an antigen on a malignant cell of interest. An inhibitory KIR-CAR(inhKIR-CAR) bearing an antigen binding moiety directed against anantigen that is present on a normal, but not malignant cell providesdampening of the activating effects from the actKIR-CAR when the T cellencounters normal cells.

In one embodiment, the present invention provides a T cell or othercytotoxic cell engineered to express at least two NKR-CARs, e.g., atleast two KIR-CARs, wherein the first NKR-CAR, e.g., a KIR-CA,R is anactNKR-CAR, e.g., an actKIR-CAR, and the second NKR-CAR, e.g., aKIR-CAR, is an inhNKR-CAR, e.g., an inhKIR-CAR. In one embodiment, theinvention provides an inhNKR-CAR, e.g., an inhKIR-CAR, wherein bindingof the inhNKR-CAR, e.g., an inhKIR-CAR, to a normal cell results ininhibition of the cytotoxic cell, e.g., inhibition of KIR-CAR T cellactivity. In one embodiment, binding of the inhNKR-CAR, e.g., aninhKIR-CAR, to an antigen associated with a non-cancerous cell resultsin the death of the NKR-CAR cytotoxic cell, e.g., a KIR-CAR T cell.

In one embodiment, an inhNKR-CAR, e.g., an actKIR-CAR, of the inventioncan be used in combination with existing CARs in order to regulate theactivity of the CARs. Exemplary CARs have been described in PCT/US11/64191, which is incorporated in its entirety by reference herein.

It has also been discovered that, in cells having a plurality ofchimeric membrane embedded receptors comprising an antigen bindingdomain (CMERs), interactions between the antigen binding domain of theCMERs can be undesirable, e.g., because interaction inhibits the abilityof one or more of the antigen binding domains to bind its cognateantigen or might generate novel binding sites with unknown cognateantigen. Accordingly, disclosed herein are cells having a first and asecond non-naturally occurring CMER wherein the antigen binding domainsminimizes such interactions. Also disclosed herein are nucleic acidsencoding a first and a second non-naturally occurring such CMERs, aswell as methods of making and using such cells and nucleic acids. In anembodiment, the antigen binding domain of one of said first said secondnon-naturally occurring CMER, comprises an scFv, and the other comprisesa single VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence or anon-antibody scaffold.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of and/or for the testing of the present invention, thepreferred materials and methods are described herein. In describing andclaiming the present invention, the following terminology will be usedaccording to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, in some instances ±5%, in some instances±1%, and in some instance ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

Adaptor molecule, as that term is used herein, refers to a polypeptidewith a sequence that permits interaction with two or more molecules, andin embodiments, promotes activation or inactivation of a cytotoxic cell.E.g., in the case of DAP12, this comprises interactions with anactivating KIR via charge interactions within the transmembrane domainand interactions with signaling molecules like ZAP70 or Syk via aphosphorylated ITAM sequence within the cytoplasmic domain.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to encodepolypeptides that elicit the desired immune response. Moreover, askilled artisan will understand that an antigen need not be encoded by a“gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as if it wereforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

CAR, as that term is used herein, refers to a chimeric polypeptide thatshares structural and functional properties with a cell immune-functionreceptor or adaptor molecule, from e.g., a T cell or a NK cell. CARsinclude TCARs and NKR-CARs. Upon binding to cognate antigen, a CAR canactivate or inactivate the cytotoxic cell in which it is disposed, ormodulate the cell's antitumor activity or otherwise modulate the cellsimmune response.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer, glioma, andthe like.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, forexample, one or more amino acid residues within the CDR regions of anantibody of the invention can be replaced with other amino acid residuesfrom the same side chain family and the altered antibody can be testedfor the ability to bind FRβ using the functional assays describedherein.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

Cytoplasmic and intracellular, as applied to adaptor molecules andsignaling domains are used interchangeably herein.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result. Such results may include, butare not limited to, the inhibition of virus infection as determined byany means suitable in the art.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

FcR-CAR, as that term is used herein, refers to a CAR which sharesfunctional and structural properties with a FcR.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Homologous” as used herein, refers to the subunit sequence identitybetween two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions; e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two sequences are homologous, the two sequences are 50%homologous; if 90% of the positions (e.g., 9 of 10), are matched orhomologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Intracellular signaling domain”, as used herein, refers to apolypeptide sequence that is a component of a larger integral membraneprotein. This polypeptide sequence, through regulated interactions withother cellular proteins, is capable of stimulating or inhibiting immunecell function such as lytic granule release, cytokine production orproliferation.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

KIR-CAR, as that term is used herein, refers to a CAR which sharesfunctional and structural properties with a KIR.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

Ly49-CAR, as that term is used herein, refers to a CAR which sharesfunctional and structural properties with a Ly49.

NCR-CAR, as that term is used herein, refers to a CAR which sharesfunctional and structural properties with a NCR.

NK cell immune-function receptor (or NKR), as that term is used herein,refers to an endogenous naturally occurring transmembrane proteinexpressed in NK cells, which can engage with a ligand on an antigenpresenting cell and modulate an NK cell immune-function response, e.g.,it can modulate the cytolytic activity or cytokine secretion of the NKcell. The NKR can contribute to activation (an activating NKR, oractNKR), or inhibition (an inhibitory NKR, or inhNKR). Typically, an NKRcomprises an extracellular ligand-binding domain (ECD), a transmembranedomain (TM) and an intracellular cytoplasmic domain (ICD). NKRs includethe Killer Immunoglobulin-like Receptor (KIR) family of receptors suchas KIR2DS2, the NK cell receptor (NCR) receptor family of receptors suchas NKp46 (NCR1), the signaling lymphocyte activation receptor (SLAM)family (SLAMF) of receptors such as 2B4, and the Fc-binding receptorssuch as the IgG-binding receptor, CD16 (FcγRIII). Examples of NK cellimmune-function responses modulated by NKRs comprise target cell killing(often referred to as cytotoxicity or cytolysis), cytokine secretionand/or proliferation. Typically, an NKR suitable for use in the methodsand compositions described herein is a human NKR, (or hNKR). In anembodiment, the Ly49 receptor family in Mus musculus, which emerged byconvergent evolution to provide the same function as a KIR in murine NKand T cells, is also included.

NKR-CAR, as that term is used herein, refers to a CAR which sharesfunctional and structural properties with a NKR or adaptor molecule froma NK cell.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

TCAR, as that term is used herein, refers to a CAR which sharesfunctional and structural properties with a cell immune-functionreceptor or adaptor molecule from a T cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. The phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal andtransmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals).

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m7G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 (SEQ ID NO:67), preferably greater than 64, more preferably greater than 100, mostpreferably greater than 300 or 400. poly(A) sequences can be modifiedchemically or enzymatically to modulate mRNA functionality such aslocalization, stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

As used herein, the term “TCAR” comprises an antigen domain, anintracellular signaling domain, and optionally one or more costimulatorydomains.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,lentiviral vectors, and the like.

By the term “specifically binds,” as used herein, is meant an antibody,or a ligand, which recognizes and binds with a cognate binding partnerprotein present in a sample, but which antibody or ligand does notsubstantially recognize or bind other molecules in the sample.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule with its cognate ligand therebymediating a signal transduction event, such as, but not limited to,signal transduction via the appropriate NK receptor.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

NKR-CARs

Disclosed herein are compositions and methods for regulating thespecificity and activity of cytotoxic cells, e.g., T cells or NK cells,e.g., with a non-naturally occurring chimeric antigen receptor (CAR). Inan embodiment the CAR is an NKR-CAR. A NKR-CAR is a CAR which sharesfunctional and structural properties with a NK cell immune-functionreceptor (or NKR). NKRs and NKR-CARs are described herein, e.g., in thesection below. As is discussed below, a variety of NKRs can serve as thebasis for an NKR-CAR.

NK Cell Immune-Function Receptors (NKRs) and NK Cells

As discussed herein, NK cell immune-function receptor (or NKR) refers toan endogenous naturally occurring transmembrane protein expressed in NKcells, which can engage with a ligand on an antigen presenting cell andmodulate an NK cell immune-function response, e.g., it can modulate thecytolytic activity or cytokine secretion of the NK cell.

NK cells are mononuclear cells that develop in the bone marrow fromlymphoid progenitors, and morphological features and biologicalproperties typically include the expression of the cluster determinants(CDs) CD16, CD56, and/or CD57; the absence of the alpha/beta orgamma/delta TCR complex on the cell surface; the ability to bind to andkill target cells that fail to express “self” major histocompatibilitycomplex (MHC)/human leukocyte antigen (HLA) proteins; and the ability tokill tumor cells or other diseased cells that express ligands foractivating NK receptors. NK cells are characterized by their ability tobind and kill several types of tumor cell lines without the need forprior immunization or activation. NK cells can also release solubleproteins and cytokines that exert a regulatory effect on the immunesystem; and can undergo multiple rounds of cell division and producedaughter cells with similar biologic properties as the parent cell. Uponactivation by interferons and/or cytokines, NK cells mediate the lysisof tumor cells and of cells infected with intracellular pathogens bymechanisms that require direct, physical contacts between the NK celland the target cell. Lysis of target cells involves the release ofcytotoxic granules from the NK cell onto the surface of the boundtarget, and effector proteins such as perforin and granzyme B thatpenetrate the target plasma membrane and induce apoptosis or programmedcell death. Normal, healthy cells are protected from lysis by NK cells.NK cell activity is regulated by a complex mechanism that involves bothstimulating and inhibitory signals.

Briefly, the lytic activity of NK cells is regulated by various cellsurface receptors that transduce either positive or negativeintracellular signals upon interaction with ligands on the target cell.The balance between positive and negative signals transmitted via thesereceptors determines whether or not a target cell is lysed (killed) by aNK cell. NK cell stimulatory signals can be mediated by NaturalCytotoxicity Receptors (NCR) such as NKp30, NKp44, and NKp46; as well asNKG2C receptors, NKG2D receptors, certain activating killer cellimmunoglobulin-like receptors (KIRs), and other activating NK receptors(Lanier, Annual Review of Immunology 2005; 23:225-74). NK cellinhibitory signals can be mediated by receptors like Ly49, CD94/NKG2A,as well as certain inhibitory KIRs, which recognize majorhistocompatibility complex (MHC) class I molecules (Karre et al., Nature1986; 319:675-8; Ohlen et al, Science 1989; 246:666-8). These inhibitoryreceptors bind to polymorphic determinants of MHC class I molecules(including HLA class I) present on other cells and inhibit NKcell-mediated lysis.

KIR-CARs

Disclosed herein is a chimeric antigen receptor (CAR) moleculecomprising an antigen binding moiety and a killer cellimmunoglobulin-like receptor (KIR-CAR). In one embodiment, the KIR-CARof the invention is expressed on the surface of a T cell.

KIR-CAR Based NKCARs

KIRs, referred to as killer cell immunoglobulin-like receptors, havebeen characterized in humans and non-human primates, and are polymorphictype 1 trans-membrane molecules present on certain subsets oflymphocytes, including NK cells and some T cells. KIRs interact withdeterminants in the alpha 1 and 2 domains of the MHC class I moleculesand, as described elsewhere herein, distinct KIRs are either stimulatoryor inhibitory for NK cells.

NKCARs described herein include KIR-CARs, which share functional andstructural properties with KIRs.

KIRs are a family of cell surface proteins found on NK cells. Theyregulate the killing function of these cells by interacting with MHCclass I molecules, which are expressed on all cell types. Thisinteraction allows them to detect virally infected cells or tumor cells.Most KIRs are inhibitory, meaning that their recognition of MHCsuppresses the cytotoxic activity of the NK cell that expresses them.Only a limited number of KIRs have the ability to activate cells.

The KIR gene family have at least 15 gene loci (KIR2DL1, KIR2DL2/L3,KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS 1, KIR2DS2, KIR2DS3, KIR2DS4,KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3 and two pseudogenes, KIR2DP1 andKIR3DP1) encoded within a 100-200 Kb region of the Leukocyte ReceptorComplex (LRC) located on chromosome 19 (19q13.4). The LRC constitutes alarge, 1 Mb, and dense cluster of rapidly evolving immune genes whichcontains genes encoding other cell surface molecules with distinctiveIg-like extra-cellular domains. In addition, the extended LRC containsgenes encoding the transmembrane adaptor molecules DAP10 and DAP12.

KIR genes vary in length from 4 to 16 Kb (full genomic sequence) and cancontain four to nine exons. KIR genes are classified as belonging to oneof three groups according to their structural features: (1) Type I KIR2Dgenes, which encode two extra-cellular domain proteins with a D1 and D2conformation; (2) The structurally divergent Type II KIR2D genes whichencode two extra-cellular domain proteins with a D0 and D2 conformation;and finally (3) KIR3D genes encoding proteins with three extra-cellularIg-like domains (D0, D1 and D2).

Type I KIR2D genes, which include the pseudogene KIR2DP1 as well asKIR2DL1-3 and KIR2DS 1-5 genes, possess eight exons as well as apseudoexon 3 sequence. This pseudoexon is inactivated in Type I KIR2D.In some cases this is due to a nucleotide substitution located on theintron 2-exon 3 splice-site where its nucleotide sequence exhibits ahigh-degree of identity to KIR3D exon 3 sequences and possesses acharacteristic three base pair deletion. In other cases a premature stopcodon initiates differential splicing of exon 3. Within the Type I KIR2Dgroup of genes, KIR2DL1 and KIR2DL2 share a common deletion in exon 7distinguishing them from all other KIR in this exon, which results in ashorter exon 7 sequence. Similarly, within Type I KIR2D, KIR2DL1-3differ from KIR2DS 1-5 only in the length of their cytoplasmic tailencoding region in exon 9. The KIR2DP1 pseudogene structure differs fromthat of KIR2DL1-3 in that the former has a shorter exon 4 sequence, dueto a single base pair deletion.

Type II KIR2D genes include KIR2DL4 and KIR2DL5. Unlike KIR3D and Type IKIR2D, Type II KIR2D characteristically have deleted the regioncorresponding to exon 4 in all other KIR. Additionally, Type II KIR2Dgenes differ from Type I KIR2D genes in that the former possess atranslated exon 3, while Type I KIR2D genes have an untranslatedpseudoexon 3 sequence in its place. Within the Type II KIR2D genes,KIR2DL4 is further differentiated from KIR2DL5 (as well as from otherKIR genes) by the length of its exon 1 sequence. In KIR2DL4, exon 1 wasfound to be six nucleotides longer and to possess an initiation codondifferent from those present in the other KIR genes. This initiationcodon is in better agreement with the ‘Kozak transcription initiationconsensus sequence’ than the second potential initiation codon inKIR2DL4 that corresponds to the initiation codon present in other KIRgenes.

KIR3D genes possess nine exons and include the structurally relatedKIR3DL1, KIR3DS 1, KIR3DL2 and KIR3DL3 genes. KIR3DL2 nucleotidesequences are the longest of all KIR genes and span 16,256 bp in fullgenomic sequences and 1,368 bp in cDNA. Within the KIR3D group, the fourKIR genes differ in the length of the region encoding the cytoplasmictail in exon 9. The length of the cytoplasmic tail of KIR proteins canvary from 14 amino acid residues long (in some KIR3DS1 alleles) to 108amino acid residues long (in KIR2DL4 proteins). Additionally, KIR3DS 1differs from KIR3DL1 or KIR3DL2 in that the former has a shorter exon 8sequence. KIR3DL3 differs from other KIR sequences in that it completelylacks exon 6. The most extreme KIR gene structure difference observedwas that of KIR3DP1. This gene fragment completely lacks exons 6 through9, and occasionally also exon 2. The remaining portions of the genewhich are present (exon 1, 3, 4 and 5) share a high level of sequenceidentity to other KIR3D sequences, in particular to KIR3DL3 sequences.

KIR proteins possess characteristic Ig-like domains on theirextracellular regions, which in some KIR proteins are involved in HLAclass I ligand binding. They also possess transmembrane and cytoplasmicregions which are functionally relevant as they define the type ofsignal which is transduced to the NK cell. KIR proteins can have two orthree Ig-like domains (hence KIR2D or KIR3D) as well as short or longcytoplasmic tails (represented as KIR2DS or KIR2DL). Two domain KIRproteins are subdivided into two groups depending on the origin of themembrane distal Ig-like domains present. Type I KIR2D proteins (KIR2DL1,KIR2DL2, KIR2DL3, KIR2DS 1, KIR2DS2, KIR2DS3, KIR2DS4 and KIR2DS5)possess a membrane-distal Ig-like domain similar in origin to the KIR3DD1 Ig-like domain but lack a D0 domain. This D1 Ig-like domain isencoded mainly by the fourth exon of the corresponding KIR genes. TheType II KIR2D proteins, KIR2DL4 and KIR2DL5, possess a membrane-distalIg-like domain of similar sequence to the D0 domain present in KIR3Dproteins, however, Type II KIR2D lack a D1 domain. Long cytoplasmictails usually contain two Immune Tyrosine-based Inhibitory Motifs (ITIM)which transduce inhibitory signals to the NK cell. Short cytoplasmictails possess a positively charged amino acid residue in theirtransmembrane region which allows them to associate with a DAP12signaling molecule capable of generating an activation signal Exceptionsto this is KIR2DL4, which contains only one N-terminus ITIM. Inaddition, KIR2DL4 also possesses a charged residue (arginine) in itstransmembrane domain, a feature which allows this receptor to elicitboth inhibitory and activating signals. KIR control the response ofhuman NK cells by delivering inhibitory or activating signals uponrecognition of MHC class I ligands on the surface of potential targetcells.

KIR proteins vary in length from 306 to 456 amino acid residues.Although the differences in protein length are mostly the consequence ofthe number of Ig-like domains present, cytoplasmic region lengthdiversity is also an influencing factor. The leader peptide of most KIRproteins is 21 amino acid residues long. However, the presence of adifferent initiation codon generates a correspondingly longer leaderpeptide in KIR2DL4 proteins.

The D0 Ig-like domain present in Type II KIR2D proteins and KIR3Dproteins is approximately 96 amino acid residues in length. The D1domain of Type I KIR2D and of KIR3D proteins is 102 amino acid residueslong, while the D2 domain of all KIR proteins is 98 amino acid residueslong. The length of the stem region varies from the 24 amino acidresidues present in most KIR proteins, to only seven amino acid residuesin the divergent KIR3DL3 protein. The transmembrane region is 20 aminoacid residues long for most KIR proteins, but one residue shorter onKIR2DL1 and KIR2DL2 proteins as a result of a three base pair deletionin exon 7. Finally, the cytoplasmic region of KIR proteins exhibitsgreater length variations, ranging from 23 amino acid residues in someKIR3DS 1 alleles to the 96 amino acid residues present in KIR3DL2proteins.

Amino acid sequences for human KIR polypeptides (Homo sapiens) areavailable in the NCBI database, see e.g., accession number NP_037421.2(GI:134268644), NP_703144.2 (GI:46488946), NP_001229796.1(GI:338968852), NP_001229796.1 (GI:338968852), NP_006728.2(GI:134268642), NP_065396.1 (GI: 11968154), NP_001018091.1(GI:66267727), NP_001077008.1 (GI:134133244), NP_036444.1 (GI:6912472),NP_055327.1 (GI:7657277), NP_056952.2 (GI:71143139), NP_036446.3 (GI:116517309), NP_001074239.1 (GI: 124107610), NP_002246.5 (GI: 124107606),NP_001074241.1 (GI: 124107604), NP_036445.1 (GI:6912474).

The nomenclature for KIRs is based upon the number of extracellulardomains (KIR2D and KIR3D having two and three extracellular Ig-domains,respectively) and whether the cytoplasmic tail is long (KIR2DL orKIR3DL) or short (KIR2DS or KIR3DS). The presence or absence of a givenKIR is variable from one NK cell to another within the NK populationpresent in a single individual. Among humans, there is also a relativelyhigh level of polymorphism of KIR genes, with certain KIR genes beingpresent in some, but not all individuals. The expression of KIR alleleson NK cells is stochastically regulated, meaning that, in a givenindividual, a given lymphocyte may express one, two, or more differentKIRs, depending on the genoptype of the individual. The NK cells of asingle individual typically express different combinations of KIRs,providing a repertoire of NK cells with different specificities for MHCclass I molecules.

Certain KIR gene products cause stimulation of lymphocyte activity whenbound to an appropriate ligand. The activating KIRs all have a shortcytoplasmic tail with a charged trans-membrane residue that associateswith an adapter molecule having an Immunoreceptor Tyrosine-basedActivation Motifs (ITAMs) which transduce stimulatory signals to the NKcell. By contrast, inhibitory KIRs have a long cytoplasmic tailcontaining Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM), whichtransduce inhibitory signals to the NK cell upon engagement of their MHCclass I ligands. The known inhibitory KIRs include members of the KIR2DLand KIR3DL subfamilies. Inhibitory KIRs having two Ig domains (KIR2DL)recognize HLA-C allotypes: KIR2DL2 (formerly designated p58.2) and theclosely related, allelic gene product KIR2DL3 both recognize “group 1”HLA-C allotypes (including HLA-Cw1, -3, -7, and -8), whereas KIR2DL1(p58.1) recognizes “group 2” HLA-C allotypes (such as HLA-Cw2, -4, -5,and -6). The recognition by KIR2DL1 is dictated by the presence of a Lysresidue at position 80 of HLA-C alleles. KIR2DL2 and KIR2DL3 recognitionis dictated by the presence of an Asn residue at position 80 in HLA-C.Importantly, the great majority of HLA-C alleles have either an Asn or aLys residue at position 80. Therefore, KIR2DL1, -2, and -3 collectivelyrecognize essentially all HLA-C allotypes found in humans. One KIR withthree Ig domains, KIR3DL1 (p70), recognizes an epitope shared by HLA-Bw4alleles. Finally, KIR3DL2 (p140), a homodimer of molecules with three Igdomains, recognizes HLA-A3 and -A11.

However, the invention should not be limited to inhibitory KIRscomprising a cytoplasmic tail containing ITIM. Rather, any inhibitoryprotein having a cytoplasmic domain that is associated with aninhibitory signal can be used in the construction of the CARs of theinvention. Non-limiting examples of an inhibitory protein include butare not limited CTLA-4, PD-1, and the like. These proteins are known toinhibit T cell activation.

Accordingly, the invention provides a KIR-CAR comprising anextracellular domain that comprises a target-specific binding elementotherwise referred to as an antigen binding domain fused to a KIR orfragment thereof. In one embodiment, the KIR is an activating KIR thatcomprises a short cytoplasmic tail that associates with an adaptermolecule having an Immunoreceptor Tyrosine-based Activation Motifs(ITAMs) which transduce stimulatory signals to the NK cell (referredelsewhere herein as actKIR-CAR). In one embodiment, the KIR is aninhibitory KIR that comprises a long cytoplasmic tail containingImmunoreceptor Tyrosine-based Inhibitory Motif (ITIM), which transduceinhibitory signals (referred elsewhere herein as inhKIR-CAR). In someinstances, it is desirable to remove the hinge region for the activatingKIRs when construction an actKIR-CAR. This is because the invention ispartly based on the discovery that an activating KIR CAR in which theKIR2DS2 hinge was removed to generate the KIR2S CAR, this KIRS2 CARexhibited enhanced cytolytic activity compared to an actKIR-CARcomprising a full length wildtype KIR2DS2.

The nucleic acid sequences coding for the desired molecules of theinvention can be obtained using recombinant methods known in the art,such as, for example by screening libraries from cells expressing thegene, by deriving the gene from a vector known to include the same, orby isolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention includes retroviral and lentiviral vectorconstructs expressing a KIR-CAR that can be directly transduced into acell. The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the gene to be expressed,and a polyA tail, typically 50-2000 bases in length. RNA so produced canefficiently transfect different kinds of cells. In one embodiment, thetemplate includes sequences for the KIR-CAR.

In an embodiment, a KIR-CAR comprises an antigen binding domain and aKIR transmembrane domain. In an embodiment, a KIR-CAR comprises anantigen binding domain and a KIR intracellular domain, e.g., an inhKIRintracellular domain.

KIR D domain, as that term is used herein, refers to a D0, D1, or D2domain of a KIR.

KIR D domain, as that term is used herein, refers to a polypeptidedomain having structural and functional properties of a D domain of aKIR.

KIR D0 domain, as that term is used herein, refers to a D0 domain of aKIR. In an embodiment the KIR D0 domain of a KIR-CAR has at least 70,80, 85, 90, 95, or 99% homology with a reference sequence, e.g., anaturally occurring KIR D0 domain or a KIR D0 domain described herein.In embodiments the KIR D0 domain of a KIR-CAR differs at no more than15, 10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring KIR D0 domain or a KIR D0 domain described herein.In embodiments the KIR D0 domain of a KIR-CAR differs at no more than 5,4, 3, 2 or 1 residue from a reference sequence, e.g., a naturallyoccurring KIR D0 domain or a KIR D0 domain described herein. Inembodiments the KIR D0 domain of a KIR-CAR does not differ from, orshares 100% homology with, a reference sequence, e.g., a naturallyoccurring KIR D0 domain or a KIR D0 domain described herein.

KIR D1 domain, as that term is used herein, refers to a polypeptidedomain having structural and functional properties of a D1 domain of aKIR. In an embodiment the KIR D1 domain of a KIR-CAR has at least 70,80, 85, 90, 95, or 99% homology with a reference sequence, e.g., anaturally occurring KIR D1 domain or a KIR D1 domain described herein.In embodiments the KIR D1 domain of a KIR-CAR differs at no more than15, 10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring KIR D1 domain or a KIR D1 domain described herein.In embodiments the KIR D1 domain of a KIR-CAR differs at no more than 5,4, 3, 2 or 1 residue from a reference sequence, e.g., a naturallyoccurring KIR D0 domain or a KIR D1 domain described herein. Inembodiments the KIR D1 domain of a KIR-CAR does not differ from, orshares 100% homology with, a reference sequence, e.g., a naturallyoccurring KIR D1 domain or a KIR D1 domain described herein.

KIR D2 domain, as that term is used herein, refers to a polypeptidedomain having structural and functional properties of a D2 domain of aKIR. In an embodiment the KIR D2 domain of a KIR-CAR has at least 70,80, 85, 90, 95, or 99% homology with a reference sequence, e.g., anaturally occurring KIR D2 domain or a KIR D2 domain described herein.In embodiments the KIR D2 domain of a KIR-CAR differs at no more than15, 10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring KIR D2 domain or a KIR D2 domain described herein.In embodiments the KIR D2 domain of a KIR-CAR differs at no more than 5,4, 3, 2 or 1 residue from a reference sequence, e.g., a naturallyoccurring KIR D2 domain or a KIR D2 domain described herein. Inembodiments the KIR D2 domain of a KIR-CAR does not differ from, orshares 100% homology with, a reference sequence, e.g., a naturallyoccurring KIR D2 domain or a KIR D0 domain described herein.

KIR hinge or stem domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of ahinge or stem domain of a KIR. In an embodiment the KIR hinge or stemdomain of a KIR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring KIR hinge or stemdomain or a KIR hinge or stem domain described herein. In embodimentsthe KIR hinge or stem domain of a KIR-CAR differs at no more than 15,10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring KIR hinge or stem domain or a KIR hinge or stemdomain described herein. In embodiments the KIR hinge or stem domain ofa KIR-CAR differs at no more than 5, 4, 3, 2 or 1 residue from areference sequence, e.g., a naturally occurring KIR hinge or stem domainor a KIR hinge or stem domain described herein. In embodiments the KIRhinge or stem domain of a KIR-CAR does not differ from, or shares 100%homology with, a reference sequence, e.g., a naturally occurring KIRhinge or stem domain or a KIR hinge or stem domain described herein.

KIR transmembrane domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of atransmembrane domain of a KIR. In an embodiment the KIR transmembranedomain of a KIR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring KIR transmembranedomain or a KIR transmembrane domain described herein. In embodimentsthe KIR transmembrane domain of a KIR-CAR differs at no more than 15,10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring KIR transmembrane domain or a KIR transmembranedomain described herein. In embodiments the KIR transmembrane domain ofa KIR-CAR differs at no more than 5, 4, 3, 2 or 1 residue from areference sequence, e.g., a naturally occurring KIR transmembrane domainor a KIR transmembrane domain described herein. In embodiments the KIRtransmembrane domain of a KIR-CAR does not differ from, or shares 100%homology with, a reference sequence, e.g., a naturally occurring KIRtransmembrane domain or a KIR transmembrane domain described herein.

KIR intracelluar domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of anintracellular domain of a KIR. KIR intracellular domains compriseinhibitory KIR intracellular domains (referred to herein as inhKIRintracellular domains) and activating KIR intracellular domains(referred to herein as actKIR intracellular domains). In an embodimentthe inhKIR intracellular domain comprises an ITIM sequence. In anembodiment the KIR intracellular domain of a KIR-CAR has at least 70,80, 85, 90, 95, or 99% homology with a reference sequence, e.g., anaturally occurring KIR intracellular domain or a KIR intracellulardomain described herein. In embodiments the KIR intracellular domain ofa KIR-CAR differs at no more than 15, 10, 5, 2, or 1% of its residuesfrom a reference sequence, e.g., a naturally occurring KIR intracellulardomain or a KIR intracellular domain described herein. In embodimentsthe KIR intracellular domain of a KIR-CAR differs at no more than 5, 4,3, 2 or 1 residue from a reference sequence, e.g., a naturally occurringKIR intracellular domain or a KIR intracellular domain described herein.In embodiments the KIR intracellular domain of a KIR-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring KIR intracellular domain or a KIR intracellulardomain described herein.

NCRs

NKCARs described herein include NCR-CARs, which share functional andstructural properties with NCRs.

Natural killer (NK) cells are cytotoxic lymphoid cells specialized indestroying tumors and virus-infected cells. Unlike cytotoxic Tlymphocytes, NK cells do not express antigen-specific receptors. Therecognition of transformed cells occurs via the association of amultitude of cell-surface receptors with surface markers on the targetcell. The NK cell surface receptors can be distinguished according towhether they activate or inhibit NK cell-mediated cytotoxicity. Numerousinteractions between different receptors appear to lead to the formationof synapses between NK and target cells. The integration of activatingand inhibiting signals at the synapse dictates whether or not the NKcells exert their cytolytic function on the target cell. Among theactivating receptors, the family of Ig-like molecules is termed naturalcytotoxicity receptors (NCRs). These natural cytotoxicity receptorsinclude NKp30, NKp44 and NKp46 molecules. The NCRs are key activatingreceptors for NK cells in tumor cell recognition. All three NCRs areinvolved in the clearance of both tumor and virus-infected cells. In thelatter, the antiviral activity is initiated by the interaction of NKp44with hemagglutinin of influenza virus or Sendai virus. NKp46 targetsvirus-infected cells by binding to influenza virus hemagglutinin orSendai virus hemagglutinin-neuraminidase. In contrast, it has been shownthat NK cell-mediated cytotoxicity is inhibited by binding of NKp30 tothe human cytomegaloviral protein pp65 (see, e.g., Arnon, et. al., Nat.Immunol. (2005) 6:515-523).

Amino acid sequences for a human NCR polypeptides (Homo sapiens) areavailable in the NCBI database, see e.g., accession number NP_004819.2(GI: 153945782), 014931.1 (GI:47605770), 095944.2 (GI:251757303),076036.1 (GI:47605775), NP_001138939.1 (GI:224586865), and/orNP_001138938.1 (GI:224586860).

In an embodiment, a NCR-CAR comprises an antigen binding domain and aNCR transmembrane domain. In an embodiment, a KIR-CAR comprises anantigen binding domain and a NCR intracellular domain.

NCR extracellular domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of aextracellular domain of a NCR. In an embodiment the NCR extracellulardomain of a NCR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring NCR extracellulardomain or a NCR extracellular domain described herein. In embodimentsthe NCR extracellular domain of a NCR-CAR differs at no more than 15,10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring NCR extracellular domain or a NCR extracellulardomain described herein. In embodiments the NCR extracellular domain ofa NCR-CAR differs at no more than 5, 4, 3, 2 or 1 residue from areference sequence, e.g., a naturally occurring NCR extracellular domainor a NCR extracellular domain described herein. In embodiments the NCRextracellular domain of a NCR-CAR does not differ from, or shares 100%homology with, a reference sequence, e.g., a naturally occurring NCRextracellular domain or a NCR extracellular domain described herein.

NCR hinge or stem domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of ahinge or stem domain of a NCR. In an embodiment the NCR hinge or stemdomain of a NCR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring NCR hinge or stemdomain or a NCR hinge or stem domain described herein. In embodimentsthe NCR hinge or stem domain of a NCR-CAR differs at no more than 15,10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring NCR hinge or stem domain or a NCR hinge or stemdomain described herein. In embodiments the NCR hinge or stem domain ofa NCR-CAR differs at no more than 5, 4, 3, 2 or 1 residue from areference sequence, e.g., a naturally occurring NCR hinge or stem domainor a NCR hinge or stem domain described herein. In embodiments the NCRhinge or stem domain of a NCR-CAR does not differ from, or shares 100%homology with, a reference sequence, e.g., a naturally occurring NCRhinge or stem domain or a NCR hinge or stem domain described herein.

NCR transmembrane domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of atransmembrane domain of a NCR. In an embodiment the NCR transmembranedomain of a NCR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring NCR transmembranedomain or a NCR transmembrane domain described herein. In embodimentsthe NCR transmembrane domain of a NCR-CAR differs at no more than 15,10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring NCR transmembrane domain or a NCR transmembranedomain described herein. In embodiments the NCR transmembrane domain ofa NCR-CAR differs at no more than 5, 4, 3, 2 or 1 residue from areference sequence, e.g., a naturally occurring NCR transmembrane domainor a NCR transmembrane domain described herein. In embodiments the NCRtransmembrane domain of a NCR-CAR does not differ from, or shares 100%homology with, a reference sequence, e.g., a naturally occurring NCRtransmembrane domain or a NCR transmembrane domain described herein.

NCR intracelluar domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of anintracellular domain of a NCR. In an embodiment the NCR intracellulardomain of a NCR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring NCR intracellulardomain or a NCR intracellular domain described herein. In embodimentsthe NCR intracellular domain of a NCR-CAR differs at no more than 15,10, 5, 2, or 1% of its residues from a reference sequence, e.g., anaturally occurring NCR intracellular domain or a NCR intracellulardomain described herein. In embodiments the NCR intracellular domain ofa NCR-CAR differs at no more than 5, 4, 3, 2 or 1 residue from areference sequence, e.g., a naturally occurring NCR intracellular domainor a NCR intracellular domain described herein. In embodiments the NCRintracellular domain of a NCR-CAR does not differ from, or shares 100%homology with, a reference sequence, e.g., a naturally occurring NCRintracellular domain or a NCR intracellular domain described herein.

SLAM Receptors

NKCARs described herein include SLAMF-CARs, which share functional andstructural properties with SLAMFs.

The signaling lymphocyte activation molecule (SLAM) family of immunecell receptors is closely related to the CD2 family of theimmunoglobulin (Ig) superfamily of molecules. The SLAM family (SLAMF)currently includes nine members named SLAM, CD48, CD229, 2B4, CD84,NTB-A, CRACC, BLAME, and CD2F-10. In general, SLAM molecules possess twoto four extracellular Ig domains, a transmembrane segment, and anintracellular tyrosine-rich region. The molecules are differentiallyexpressed on a variety of immune cell types. Several are self ligandsand SLAM has been identified as the human measles virus receptor.Several small SH2-containing adaptor proteins are known to associatewith the intracellular domains of SLAM family members and modulatereceptor signaling including SH2D1A (also known as SLAM-associatedprotein [SAP]) and SH2D1B (also known as EAT2). For example, in T and NKcells, activated SLAM family receptors become tyrosine phosphorylatedand recruit the adaptor SAP and subsequently the Src kinase Fyn. Theensuing signal transduction cascade influences the outcome of Tcell-antigen presenting cell and NK cell-target cell interactions.

Amino acid sequences for human SLAM receptor polypeptides (Homo sapiens)are available in the NCBI database, see e.g., accession numberNP_057466.1 (GI: 7706529), NP_067004.3 (GI: 19923572), NP_003028.1(GI:4506969), NP_001171808.1 (GI: 296434285), NP_001171643.1(GI:296040491), NP_001769.2 (GI:21361571), NP_254273.2 (GI: 226342990),NP_064510.1 (GI: 9910342) and/or NP_002339.2 (GI: 55925578)

In an embodiment, a SLAMF-CAR comprises an antigen binding domain and aSLAMF transmembrane domain. In an embodiment, a SLAMF-CAR comprises anantigen binding domain and a NCR intracellular domain.

SLAMF extracellular domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of aextracellular domain of a SLAMF. In an embodiment the SLAMFextracellular domain of a SLAMF-CAR has at least 70, 80, 85, 90, 95, or99% homology with a reference sequence, e.g., a naturally occurringSLAMF extracellular domain or a SLAMF extracellular domain describedherein. In embodiments the SLAMF extracellular domain of a SLAMF-CARdiffers at no more than 15, 10, 5, 2, or 1% of its residues from areference sequence, e.g., a naturally occurring SLAMF extracellulardomain or a SLAMF extracellular domain described herein. In embodimentsthe SLAMF extracellular domain of a SLAMF-CAR differs at no more than 5,4, 3, 2 or 1 residue from a reference sequence, e.g., a naturallyoccurring SLAMF extracellular domain or a SLAMF extracellular domaindescribed herein. In embodiments the SLAMF extracellular domain of aSLAMF-CAR does not differ from, or shares 100% homology with, areference sequence, e.g., a naturally occurring SLAMF extracellulardomain or a SLAMF extracellular domain described herein.

SLAMF hinge or stem domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of ahinge or stem domain of a SLAMF. In an embodiment the SLAMF hinge orstem domain of a SLAMF-CAR has at least 70, 80, 85, 90, 95, or 99%homology with a reference sequence, e.g., a naturally occurring SLAMFhinge or stem domain or a SLAMF hinge or stem domain described herein.In embodiments the SLAMF hinge or stem domain of a SLAMF-CAR differs atno more than 15, 10, 5, 2, or 1% of its residues from a referencesequence, e.g., a naturally occurring SLAMF hinge or stem domain or aSLAMF hinge or stem domain described herein. In embodiments the SLAMFhinge or stem domain of a SLAMF-CAR differs at no more than 5, 4, 3, 2or 1 residue from a reference sequence, e.g., a naturally occurringSLAMF hinge or stem domain or a SLAMF hinge or stem domain describedherein. In embodiments the SLAMF hinge or stem domain of a SLAMF-CARdoes not differ from, or shares 100% homology with, a referencesequence, e.g., a naturally occurring SLAMF hinge or stem domain or aSLAMF hinge or stem domain described herein.

SLAMF transmembrane domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of atransmembrane domain of a SLAMF. In an embodiment the SLAMFtransmembrane domain of a SLAMF-CAR has at least 70, 80, 85, 90, 95, or99% homology with a reference sequence, e.g., a naturally occurringSLAMF transmembrane domain or a SLAMF transmembrane domain describedherein. In embodiments the SLAMF transmembrane domain of a SLAMF-CARdiffers at no more than 15, 10, 5, 2, or 1% of its residues from areference sequence, e.g., a naturally occurring SLAMF transmembranedomain or a SLAMF transmembrane domain described herein. In embodimentsthe SLAMF transmembrane domain of a SLAMF-CAR differs at no more than 5,4, 3, 2 or 1 residue from a reference sequence, e.g., a naturallyoccurring SLAMF transmembrane domain or a SLAMF transmembrane domaindescribed herein. In embodiments the SLAMF transmembrane domain of aSLAMF-CAR does not differ from, or shares 100% homology with, areference sequence, e.g., a naturally occurring SLAMF transmembranedomain or a SLAMF transmembrane domain described herein.

SLAMF intracelluar domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of anintracellular domain of a SLAMF. In an embodiment the SLAMFintracellular domain of a SLAMF-CAR has at least 70, 80, 85, 90, 95, or99% homology with a reference sequence, e.g., a naturally occurringSLAMF intracellular domain or a SLAMF intracellular domain describedherein. In embodiments the SLAMF intracellular domain of a SLAMF-CARdiffers at no more than 15, 10, 5, 2, or 1% of its residues from areference sequence, e.g., a naturally occurring SLAMF intracellulardomain or a SLAMF intracellular domain described herein. In embodimentsthe SLAMF intracellular domain of a SLAMF-CAR differs at no more than 5,4, 3, 2 or 1 residue from a reference sequence, e.g., a naturallyoccurring SLAMF intracellular domain or a SLAMF intracellular domaindescribed herein. In embodiments the SLAMF intracellular domain of aSLAMF-CAR does not differ from, or shares 100% homology with, areference sequence, e.g., a naturally occurring SLAMF intracellulardomain or a SLAMF intracellular domain described herein.

Fc-Binding Receptors

NKCARs described herein include CARs based on the Fc receptors,FcR-CARs, e.g., CD16-CARs, and CD64-CARs, which share functional andstructural properties with CD16 and CD64.

Upon activation, NK cells produce cytokines and chemokines abundantlyand at the same time exhibit potent cytolytic activity. Activation of NKcells can occur through the direct binding of NK cell receptors toligands on the target cell, as seen with direct tumor cell killing, orthrough the crosslinking of the Fc receptor (CD 16; FcγRIII) by bindingto the Fc portion of antibodies bound to an antigen-bearing cell. ThisCD16 engagement (CD16 crosslinking) initiates NK cell responses viaintracellular signals that are generated through one, or both, of theCD16-associated adaptor chains, FcRγ or CD3 ζ. Triggering of CD16 leadsto phosphorylation of the γ or ζ chain, which in turn recruits tyrosinekinases, syk and ZAP-70, initiating a cascade of signal transductionleading to rapid and potent effector functions. The most well-knowneffector function is the release of cytoplasmic granules carrying toxicproteins to kill nearby target cells through the process ofantibody-dependent cellular cytotoxicity. CD16 crosslinking also resultsin the production of cytokines and chemokines that, in turn, activateand orchestrate a series of immune responses.

However, unlike T and B lymphocytes, NK cells are thought to have only alimited capacity for target recognition using germline-encodedactivation receptors (Bottino et al., Curr Top Microbiol Immunol.298:175-182 (2006); Stewart et al., Curr Top Microbiol Immunol. 298:1-21(2006)). NK cells express the activating Fc receptor CD 16, whichrecognizes IgG-coated target cells, thereby broadening targetrecognition (Ravetch & Bolland, Annu Rev Immunol. 19:275-290 (2001);Lanier Nat. Immunol. 9(5):495-502 (2008); Bryceson & Long, Curr OpinImmunol. 20(3):344-352 (2008)).

The expression and signal transduction activity of several NK cellactivation receptors requires physically associated adaptors, whichtransduce signals through immunoreceptor tyrosine-based activationmotifs (ITAMs). Among these adaptors, FcRγ and CD3 ζ chains canassociate with CD16 and natural cytotoxicity receptors (NCRs) as eitherdisulfide-linked homo-dimers or hetero-dimers, and these chains havebeen thought to be expressed by all mature NK cells.

Amino acid sequence for CD16 (Homo sapiens) is available in the NCBIdatabase, see e.g., accession number NP_000560.5 (GI: 50726979),NP_001231682.1 (GI: 348041254)

In an embodiment, a FcR-CAR comprises an antigen binding domain and aFcR transmembrane domain. In an embodiment, a FcR-CAR comprises anantigen binding domain and a FcR intracellular domain.

CD16 extracellular domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of aextracellular domain of a CD16. In an embodiment the CD16 extracellulardomain of a CD16-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CD16extracellular domain or a CD16 extracellular domain described herein. Inembodiments the CD16 extracellular domain of a CD16-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring CD16 extracellular domain or a CD16extracellular domain described herein. In embodiments the CD16extracellular domain of a CD16-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring CD16extracellular domain or a CD16 extracellular domain described herein. Inembodiments the CD16 extracellular domain of a CD16-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring CD16 extracellular domain or a CD16 extracellulardomain described herein.

CD16 hinge or stem domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of ahinge or stem domain of a CD16. In an embodiment the CD16 hinge or stemdomain of a CD16-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CD16 hinge orstem domain or a CD16 hinge or stem domain described herein. Inembodiments the CD16 hinge or stem domain of a CD16-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring CD16 hinge or stem domain or a CD16 hinge orstem domain described herein. In embodiments the CD16 hinge or stemdomain of a CD16-CAR differs at no more than 5, 4, 3, 2 or 1 residuefrom a reference sequence, e.g., a naturally occurring CD16 hinge orstem domain or a CD16 hinge or stem domain described herein. Inembodiments the CD16 hinge or stem domain of a CD16-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring CD16 hinge or stem domain or a CD16 hinge or stemdomain described herein.

CD16 transmembrane domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of atransmembrane domain of a CD16. In an embodiment the CD16 transmembranedomain of a CD16-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CD16transmembrane domain or a CD16 transmembrane domain described herein. Inembodiments the CD16 transmembrane domain of a CD16-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring CD16 transmembrane domain or a CD16transmembrane domain described herein. In embodiments the CD16transmembrane domain of a CD16-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring CD16transmembrane domain or a CD16 transmembrane domain described herein. Inembodiments the CD16 transmembrane domain of a CD16-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring CD16 transmembrane domain or a CD16 transmembranedomain described herein.

CD16 intracelluar domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of anintracellular domain of a CD16. In an embodiment the CD16 intracellulardomain of a CD16-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CD16intracellular domain or a CD16 intracellular domain described herein. Inembodiments the CD16 intracellular domain of a CD16-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring CD16 intracellular domain or a CD16intracellular domain described herein. In embodiments the CD16intracellular domain of a CD16-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring CD16intracellular domain or a CD16 intracellular domain described herein. Inembodiments the CD16 intracellular domain of a CD16-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring CD16 intracellular domain or a CD16 intracellulardomain described herein.

CD64 extracellular domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of aextracellular domain of a CD64. In an embodiment the CD64 extracellulardomain of a CD64-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CD64extracellular domain or a CD64 extracellular domain described herein. Inembodiments the CD64 extracellular domain of a CD64-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring CD64 extracellular domain or a CD64extracellular domain described herein. In embodiments the CD64extracellular domain of a CD64-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring CD64extracellular domain or a CD64 extracellular domain described herein. Inembodiments the CD64 extracellular domain of a CD64-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring CD64 extracellular domain or a CD64 extracellulardomain described herein.

CD64 hinge or stem domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of ahinge or stem domain of a CD64. In an embodiment the CD64 hinge or stemdomain of a CD64-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CD64 hinge orstem domain or a CD64 hinge or stem domain described herein. Inembodiments the CD64 hinge or stem domain of a CD64-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring CD64 hinge or stem domain or a CD64 hinge orstem domain described herein. In embodiments the CD64 hinge or stemdomain of a CD64-CAR differs at no more than 5, 4, 3, 2 or 1 residuefrom a reference sequence, e.g., a naturally occurring CD64 hinge orstem domain or a CD64 hinge or stem domain described herein. Inembodiments the CD64 hinge or stem domain of a CD64-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring CD64 hinge or stem domain or a CD64 hinge or stemdomain described herein.

CD64 transmembrane domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of atransmembrane domain of a CD64. In an embodiment the CD64 transmembranedomain of a CD64-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CD64transmembrane domain or a CD64 transmembrane domain described herein. Inembodiments the CD64 transmembrane domain of a CD64-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring CD64 transmembrane domain or a CD64transmembrane domain described herein. In embodiments the CD64transmembrane domain of a CD64-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring CD64transmembrane domain or a CD64 transmembrane domain described herein. Inembodiments the CD64 transmembrane domain of a CD64-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring CD64 transmembrane domain or a CD64 transmembranedomain described herein. CD64 intracelluar domain, as that term is usedherein, refers to a polypeptide domain having structural and functionalproperties of an intracellular domain of a CD64. In an embodiment theCD64 intracellular domain of a CD64-CAR has at least 70, 80, 85, 90, 95,or 99% homology with a reference sequence, e.g., a naturally occurringCD64 intracellular domain or a CD64 intracellular domain describedherein. In embodiments the CD64 intracellular domain of a CD64-CARdiffers at no more than 15, 10, 5, 2, or 1% of its residues from areference sequence, e.g., a naturally occurring CD64 intracellulardomain or a CD64 intracellular domain described herein. In embodimentsthe CD64 intracellular domain of a CD64-CAR differs at no more than 5,4, 3, 2 or 1 residue from a reference sequence, e.g., a naturallyoccurring CD64 intracellular domain or a CD64 intracellular domaindescribed herein. In embodiments the CD64 intracellular domain of aCD64-CAR does not differ from, or shares 100% homology with, a referencesequence, e.g., a naturally occurring CD64 intracellular domain or aCD64 intracellular domain described herein.

Ly49 and Related Killer Cell Lectin-Like Receptors

NKCARs described herein include Ly49-CARs, which share functional andstructural properties with Ly49.

The Ly49 receptors derive from at least 23 identified genes (Ly49A-W) inmice. These receptors share many of the same roles in mouse NK cells andT cells as that played by KIRs in humans despite their differentstructure (type II integral membrane proteins of the C-type lectinsuperfamily), and they also contain a considerable degree of geneticvariation like human KIRs. The remarkable functional similarity betweenLy49 and KIR receptors suggest that these groups of receptors haveevolved independently yet convergently to perform the same physiologicfunctionals in NK cells and T cells.

Like KIRs in humans, different Ly49 receptors recognize different MHCclass I alleles and are differentially expressed on subsets of NK cells.The original prototypic Ly49 receptors, Ly49A and Ly49C possess acytoplasmic domain bearing two immunotyrosine-based inhibitory motifs(ITIM) similar to inhibitory KIRs such as KIR2DL3. These domains havebeen identified to recruit the phosphatase, SHP-1, and like theinhibitory KIRs, serve to limit the activation of NK cells and T cells.In addition to the inhibitory Ly49 molecules, several family memberssuch as Ly49D and Ly49H have lost the ITIM-containing domains, and haveinstead acquired the capacity to interact with the signaling adaptormolecule, DAP12 similar to the activating KIRs such as KIR2DS2 inhumans.

Amino acid sequence for Ly49 family members are available in the NCBIdatabase, see e.g., accession numbers AAF82184.1 (GI: 9230810),AAF99547.1 (GI: 9801837), NP_034778.2 (GI: 133922593), NP_034779.1 (GI:6754462), NP_001095090.1 (GI: 197333718), NP_034776.1 (GI: 21327665),AAK11559.1 (GI: 13021834) and/or NP_038822.3 (GI: 9256549).

In an embodiment, a Ly49-CAR comprises an antigen binding domain and aLy49 transmembrane domain. In an embodiment, a Ly49-CAR comprises anantigen binding domain and a NCR intracellular domain.

LY49 extracellular domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of aextracellular domain of a LY49. In an embodiment the LY49 extracellulardomain of a LY49-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring LY49extracellular domain or a LY49 extracellular domain described herein. Inembodiments the LY49 extracellular domain of a LY49-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring LY49 extracellular domain or a LY49extracellular domain described herein. In embodiments the LY49extracellular domain of a LY49-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring LY49extracellular domain or a LY49 extracellular domain described herein. Inembodiments the LY49 extracellular domain of a LY49-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring LY49 extracellular domain or a LY49 extracellulardomain described herein.

LY49 hinge or stem domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of ahinge or stem domain of a LY49. In an embodiment the LY49 hinge or stemdomain of a LY49-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring LY49 hinge orstem domain or a LY49 hinge or stem domain described herein. Inembodiments the LY49 hinge or stem domain of a LY49-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring LY49 hinge or stem domain or a LY49 hinge orstem domain described herein. In embodiments the LY49 hinge or stemdomain of a LY49-CAR differs at no more than 5, 4, 3, 2 or 1 residuefrom a reference sequence, e.g., a naturally occurring LY49 hinge orstem domain or a LY49 hinge or stem domain described herein. Inembodiments the LY49 hinge or stem domain of a LY49-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring LY49 hinge or stem domain or a LY49 hinge or stemdomain described herein.

LY49 transmembrane domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of atransmembrane domain of a LY49. In an embodiment the LY49 transmembranedomain of a LY49-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring LY49transmembrane domain or a LY49 transmembrane domain described herein. Inembodiments the LY49 transmembrane domain of a LY49-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring LY49 transmembrane domain or a LY49transmembrane domain described herein. In embodiments the LY49transmembrane domain of a LY49-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring LY49transmembrane domain or a LY49 transmembrane domain described herein. Inembodiments the LY49 transmembrane domain of a LY49-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring LY49 transmembrane domain or a LY49 transmembranedomain described herein.

LY49 intracelluar domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of anintracellular domain of a LY49. In an embodiment the LY49 intracellulardomain of a LY49-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring LY49intracellular domain or a LY49 intracellular domain described herein. Inembodiments the LY49 intracellular domain of a LY49-CAR differs at nomore than 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring LY49 intracellular domain or a LY49intracellular domain described herein. In embodiments the LY49intracellular domain of a LY49-CAR differs at no more than 5, 4, 3, 2 or1 residue from a reference sequence, e.g., a naturally occurring LY49intracellular domain or a LY49 intracellular domain described herein. Inembodiments the LY49 intracellular domain of a LY49-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring LY49 intracellular domain or a LY49 intracellulardomain described herein.

Intracelluar Signaling Domains or Adaptor Molecules, e.g., DAP12

Some NKR-CARs interact with other molecules, e.g., molecules comprisingan intracellular signaling domain, e.g., an ITAM. In an embodiment aintracellular signaling domain is DAP12.

DAP12 is so named because of its structural features, and presumedfunction. Certain cell surface receptors lack intrinsic functionality,which hypothetically may interact with another protein partner,suggested to be a 12 kD protein. The mechanism of the signaling mayinvolve an ITAM signal.

The DAP12 was identified from sequence databases based upon ahypothesized relationship to CD3 (see Olcese, et al. (1997) J. Immunol.158:5083-5086), the presence of an ITAM sequence (see Thomas (1995) J.Exp. Med. 181:1953-1956), certain size predictions (see Olcese; andTakase, et al. (1997) J. Immunol. 159:741-747, and other features. Inparticular, the transmembrane domain was hypothesized to contain acharged residue, which would allow a salt bridge with the correspondingtransmembrane segments of its presumed receptor partners, KIR CD94protein, and possibly other similar proteins. See Daeron, et al. (1995)Immunity 3:635-646.

In fact, many of the known KIR, MIR, ILT, and CD94/NKG2 receptormolecules may actually function with an accessory protein which is partof the functional receptor complex. See Olcese, et al. (1997) J.Immunol. 158:5083-5086; and Takase, et al. (1997) J. Immunol.159:741-747.

A DAP 12 domain, as that term is used herein, refers to a polypeptidedomain having structural and functional properties of a cytoplasmicdomain of a DAP 12, and will typically include an ITAM domain. In anembodiment a DAP 12 domain of a KIR-CAR has at least 70, 80, 85, 90, 95,or 99% homology with a reference sequence, e.g., a naturally occurringDAP 12 or a DAP 12 described herein. In embodiments the DAP 12 domain ofa KIR-CAR differs at no more than 15, 10, 5, 2, or 1% of its residuesfrom a reference sequence, e.g., a naturally occurring DAP 12 or a DAP12 described herein. In embodiments the DAP 12 domain of a KIR-CARdiffers at no more than 5, 4, 3, 2 or 1 residue from a referencesequence, e.g., a naturally occurring DAP 12 or a DAP 12 describedherein. In embodiments the DAP 12 domain of a KIR-CAR does not differfrom, or shares 100% homology with, a reference sequence, e.g., anaturally occurring DAP 12 or a DAP 12 described herein.

The DAP10 was identified partly by its homology to the DAP12, and otherfeatures. In particular, in contrast to the DAP12, which exhibits anITAM activation motif, the DAP10 exhibits an ITIM inhibitory motif. TheMDL-1 was identified by its functional association with DAP12.

The functional interaction between, e.g., DAP12 or DAP10, and itsaccessory receptor may allow use of the structural combination inreceptors which normally are not found in a truncated receptor form.Thus, the mechanism of signaling through such accessory proteins as theDAP12 and DAP10 allow for interesting engineering of other KIR-likereceptor complexes, e.g., with the KIR, MIR, ILT, and CD94 NKG2 typereceptors. Truncated forms of intact receptors may be constructed whichinteract with a DAP12 or DAP10 to form a functional signaling complex.

The primate nucleotide sequence of DAP12 corresponds to SEQ ID NO: 6;the amino acid sequence corresponds to SEQ ID NO: 7. The signal sequenceappears to run from met(−26) to gln(−1) or ala1; the mature proteinshould run from about ala1 (or gln2), the extracellular domain fromabout ala1 to pro 14; the extracellular domain contains two cysteines at7 and 9, which likely allow disulfide linkages to additional homotypicor heterotypic accessory proteins; the transmembrane region runs fromabout gly15 or val16 to about gly39; and an ITAM motif from tyr65 toleu79 (YxxL-6/8x-YxxL (SEQ ID NO: 68)). The LVA03A EST was identifiedand used to extract other overlapping sequences. See also Genbank HumanESTs that are part of human DAP12; some, but not all, inclusive GenbankAccession # AA481924; H39980; W60940; N41026; R49793; W60864; W92376;H12338; T52100; AA480109; H12392; W74783; and T55959

Inhibitory NKR-CARs

The present invention provides compositions and methods for limiting thedepletion of non-cancerous cells by a type of CAR T cell therapy. Asdisclosed herein, a type of CAR T cell therapy comprises the use of NKreceptors including but is not limited to activating and inhibitoryreceptors of NK cells known as killer cell immunoglobulin-like receptor(KIR). Accordingly the invention provides compositions and methods ofusing a NKR-CAR, e.g., a KIR-CAR, including but is not limited to anactivating NKR-CAR (actNKR-CAR), e.g., an activating KIR-CAR(actKIR-CAR) and an inhibitory NKR-CAR (inhNKR-CAR), e.g., an inhibitoryKIR-CAR (inhKIR-CAR).

In some embodiments, the KIR of an inhKIR-CARs is an inhibitory KIR thatcomprises a long cytoplasmic tail containing ImmunoreceptorTyrosine-based Inhibitory Motif (ITIM), which transduce inhibitorysignals (referred elsewhere herein as inhKIR-CAR).

In some embodiments, an inhKIR-CAR comprises a cytoplasmic domain of aninhibitory molecule other than KIR. These inhibitory molecules can, insome embodiments, decrease the ability of a cell to mount an immuneeffector response. Cytoplasmic domains of inhibitory molecules may becoupled, e.g., by fusion, to transmembrane domains of KIR. Exemplaryinhibitory molecules are shown in table 1:

TABLE 1 Inhibitory molecules CD160 2B4 PD1 TIM3 LAG3 TIGIT CTLA-4 BTLALAIR1 PD-L1 VISTA

In some embodiments, an inhKIR-CAR comprises a PD1 cytoplasmic domain. APD1 cytoplasmic domain, as that term is used herein, refers to apolypeptide domain having structural and functional properties of acytoplasmic domain of a PD 1. In an embodiment the PD1 cytoplasmicdomain of a KIR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring PD1 cytoplasmicdomain or a PD1 cytoplasmic domain described herein (SEQ ID NO: 14). Inembodiments the PD1 cytoplasmic domain of a KIR-CAR differs at no morethan 15, 10, 5, 2, or 1% of its residues from a reference sequence,e.g., a naturally occurring PD1 cytoplasmic domain or a PD1 cytoplasmicdomain described herein. In embodiments the PD1 cytoplasmic domain of aKIR-CAR differs at no more than 5, 4, 3, 2 or 1 residue from a referencesequence, e.g., a naturally occurring PD1 cytoplasmic domain or a PD1cytoplasmic domain described herein. In embodiments the PD1 cytoplasmicdomain of a KIR-CAR does not differ from, or shares 100% homology with,a reference sequence, e.g., a naturally occurring PD 1 cytoplasmicdomain or a PD 1 cytoplasmic domain described herein.

In some embodiments, an inhKIR-CAR comprises a CTLA-4 cytoplasmicdomain. A CTLA-4 cytoplasmic domain, as that term is used herein, refersto a polypeptide domain having structural and functional properties of acytoplasmic domain of a CTLA-4. In an embodiment the CTLA-4 cytoplasmicdomain of a KIR-CAR has at least 70, 80, 85, 90, 95, or 99% homologywith a reference sequence, e.g., a naturally occurring CTLA-4cytoplasmic domain or a CTLA-4 cytoplasmic domain described herein (SEQID NO: 15). In embodiments the CTLA-4 cytoplasmic domain of a KIR-CARdiffers at no more than 15, 10, 5, 2, or 1% of its residues from areference sequence, e.g., a naturally occurring CTLA-4 cytoplasmicdomain or a CTLA-4 cytoplasmic domain described herein. In embodimentsthe CTLA-4 cytoplasmic domain of a KIR-CAR differs at no more than 5, 4,3, 2 or 1 residue from a reference sequence, e.g., a naturally occurringCTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain describedherein. In embodiments the CTLA-4 cytoplasmic domain of a KIR-CAR doesnot differ from, or shares 100% homology with, a reference sequence,e.g., a naturally occurring CTLA-4 cytoplasmic domain or a CTLA-4cytoplasmic domain described herein.

In an embodiment, an inhNKR-CAR, e.g., an inhKIR-CAR, upon engagementwith an antigen on a non-target or bystander cell, inactivates thecytotoxic cell comprising the inhNKR-CAR. While much of the descriptionbelow relates to inhKIR-CARs, the invention includes the analogousapplication of other inhNKR-CARs.

In one embodiment, T cells expressing the actKIR-CAR exhibit anantitumor property when bound to its target, whereas T cells expressingan inhKIR-CAR results in inhibition of cell activity when the inhKIR-CARis bound to its target.

Regardless of the type of KIR-CAR, KIR-CARs are engineered to comprisean extracellular domain having an antigen binding domain fused to acytoplasmic domain. In one embodiment, KIR-CARs, when expressed in a Tcell, are able to redirect antigen recognition based upon the antigenspecificity. An exemplary antigen is CD19 because this antigen isexpressed on B cell lymphoma. However, CD19 is also expressed on normalB cells, and thus CARs comprising an anti-CD19 domain may result indepletion of normal B cells. Depletion of normal B cells can make atreated subject susceptible to infection, as B cells normally aid Tcells in the control of infection. The present invention provides forcompositions and methods to limit the depletion of normal tissue duringKIR-CAR T cell therapy. In one embodiment, the present inventionprovides methods to treat cancer and other disorders using KIR-CAR Tcell therapy while limiting the depletion of healthy bystander cells.

In one embodiment, the invention comprises controlling or regulatingKIR-CAR T cell activity. In one embodiment, the invention comprisescompositions and methods related to genetically modifying T cells toexpress a plurality of types of KIR-CARs, where KIR-CAR T cellactivation is dependent on the binding of a plurality of types ofKIR-CARs to their target receptor. Dependence on the binding of aplurality of types of KIR-CARs improves the specificity of the lyticactivity of the KIR-CAR T cell, thereby reducing the potential fordepleting normal healthy tissue.

In another embodiment, the invention comprises compositions and methodsrelated to genetically modifying T cells with an inhibitory KIR-CAR. Inone embodiment, the inhibitory KIR-CAR comprises an extracellularantigen binding domain that recognizes an antigen associated with anormal, non-cancerous, cell and an inhibitory cytoplasmic domain.

In one embodiment, the invention provides a dual KIR-CAR where a T cellis genetically modified to express an inhKIR-CAR and an actKIR-CAR. Inone embodiment, binding of the inhKIR-CAR to a normal, non-cancerouscell results in the inhibition of the dual KIR-CAR T cell. For example,in one embodiment, binding of the inhKIR-CAR to a normal, non-cancerouscell results in the death of the dual KIR-CAR T cell. In anotherembodiment, binding of the inhKIR-CAR to a normal, non-cancerous cellresults in inhibiting the signal transduction of the actKIR-CAR. In yetanother embodiment, binding of the inhKIR-CAR to a normal, non-cancerouscell results in the induction of a signal transduction signal thatprevents the actKIR-CAR T cell from exhibiting its anti-tumor activity.Accordingly, the dual KIR-CAR comprising at least one inhKIR-CAR and atleast one actKIR-CAR of the invention provides a mechanism to regulatethe activity of the dual KIR-CAR T cell.

In one embodiment, the present invention provides methods for treatingcancer and other disorders using KIR-CAR T cell therapies whileminimizing the depletion of normal healthy tissue. The cancer may be ahematological malignancy, a solid tumor, a primary or a metastasizingtumor. Other diseases treatable using the compositions and methods ofthe invention include viral, bacterial and parasitic infections as wellas autoimmune diseases.

Extracellular Hinge Domain

Extracellular hinge domain, as that term is used herein, refers to apolypeptide sequence of a NKCAR disposed between the transmembranedomain and antigen binding domain. In an embodiment the extracellularhinge domain allows sufficient distance from the outer surface of thecell and the antigen binding domain as well as flexibility to minimizesteric hinderance between the cell and the antigen binding domain. In anembodiment the extracellular hinge domain is sufficiently short orflexible that it does not interfere with engagement of the cell thatincludes the NKCAR with an antigen bearing cell, e.g., a target cell. Inan embodiment the extracellular hinge domain is from 2 to 20, 5 to 15, 7to 12, or 8 to 10 amino acids in length. In an embodiment the hingedomain includes at least 50, 20, or 10 residues. In embodiments thehinge is 10 to 300, 10 to 250, or 10 t 200 residues in length. In anembodiment the distance from which the hinge extends from the cell issufficiently short that the hinge does not hinder engagement with thesurface of a target cell. In an embodiment the hinge extends less than20, 15, or 10 nanometers from the surface of the cytotoxic cell. Thus,suitability for a hinge can be influenced by both linear length, thenumber of amino acid residues and flexibility of the hinge. An IgG4hinge can be as long as 200 amino acids in length, but the distance itextends from the surface of the cytotoxic cell is smaller due toIg-domain folding. A CD8alpha hinge, which is ˜43 amino acids is ratherlinear at ˜8 nm in length. In contrast, the IgG4 C2 & C3 hinge) is ˜200amino acids in length, but has a distance from the cytotoxic cellsurface comparable to that of the CD8 alpha hinge. While not wishing tobe bound by theory, the similarity in extension is influenced byflexibility.

In some instances, the extracellular hinge domain is, e.g., a hinge froma human protein, a fragment thereof, or a short oligo- or polypeptidelinker.

In some embodiments, the hinge is an artificial sequence. In oneembodiment, the hinge is a short oligopeptide linker comprising aglycine-serine doublet.

In some embodiments, the hinge is a naturally occurring sequence. Insome embodiments, the hinge can be a human Ig (immunoglobulin) hinge, orfragment thereof. In one embodiment, for example, the hinge comprises(e.g., consists of) the amino acid sequence of the IgG4 hinge (SEQ IDNO: 49). In one embodiment, for example, the hinge comprises (e.g.,consists of) the amino acid sequence of the IgD hinge (SEQ ID NO: 50).In some embodiments, the hinge can be a human CD8 hinge, or fragmentthereof. In one embodiment, for example, the hinge comprises (e.g.,consists of) the amino acid sequence of the CD8 hinge (SEQ ID NO: 51).

TCARS

In some embodiments, the CAR cell therapy of the present inventioncomprises NKR-CAR in combination with a TCAR. In one embodiment, a TCARcomprises an antigen binding domain fused to an intracellular domain. Inembodiments, an intracellular signaling domain produces an intracellularsignal when an extracellular domain, e.g., an antigen binding domain, towhich it is fused binds a counter ligand. Intracellular signalingdomains can include primary intracellular signaling domains andcostimulatory signaling domains. In an embodiment, a TCAR molecule canbe constructed for expression in an immune cell, e.g., a T cell, suchthat the TCAR molecule comprises a domain, e.g., a primary intracellularsignaling domains, costimulatory signaling domain, inhibitory domains,etc., that is derived from a polypeptide that is typically associatedwith the immune cell. For example, a TCAR for expression in a T cell cancomprise a 41BB domain and an CD3 zeta domain. In this instance, boththe 41BB and CD3 zeta domains are derived from polypeptides associatedwith the T cell. In another embodiment, a TCAR molecule can beconstructed for expression in an immune cell e.g., a T cell, such thatthe TCAR molecule comprises a domain that is derived from a polypeptidethat is not typically associated with the immune cell. Alternatively, aTCAR for expression in a NK cell can comprise a 41BB domain and a CD3zeta domain derived from a T cell (See e.g. WO2013/033626, incorporatedherein by reference).

Primary Intracelluar Signaling Domain

In some embodiments a primary intracellular signaling domain produces anintracellular signal when an extracellular domain, e.g., an antigenbinding domain, to which it is fused binds cognate antigen. It isderived from a primary stimulatory molecule, e.g., it comprisesintracellular sequence of a primary stimulatory molecule. It comprisessufficient primary stimulatory molecule sequence to produce anintracellular signal, e.g., when an antigen binding domain to which itis fused binds cognate antigen.

A primary stimulatory molecule, is a molecule, that upon binding cognateligand, mediates an immune effector response, e.g., in the cell in whichit is expressed. Typically, it generates an intracellular signal that isdependent on binding to a cognate ligand that comprises antigen. TheTCR/CD3 complex is an exemplary primary stimulatory molecule; itgenerates an intracellular signal upon binding to cognate ligand, e.g.,an MHC molecule loaded with a peptide. Typically, e.g., in the case ofthe TCR/CD3 primary stimulatory molecule, the generation of anintracellular signal by a primary intracellular signaling domain isdependent on binding of the primary stimulatory molecule to antigen.

Primary stimulation can mediate altered expression of certain molecules,such as downregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like. Stimulation, can, e.g., in the presence ofcostimulation, result in an optimization, e.g., an increase, in animmune effector function of the T cell. Stimulation, e.g., in thecontext of a T cell, can mediate a T cell response, e.g., proliferation,activation, differentiation, and the like.

In an embodiment, the primary intracellular signaling domain comprises asignaling motif, e.g., an immunoreceptor tyrosine-based activation motifor ITAMs. A primary intracellular signaling domain can comprise ITAMcontaining cytoplasmic signaling sequences from TCR zeta (CD3 zeta), FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, CD278 (also known as “ICOS”) and CD66d.

Exemplary primary intracellular signaling domains are provided in Table2.

TABLE 2 Primary Intracellular Signaling Domains In embodiments thedomain comprises an ITAM TCR zeta FcR gamma FcR beta CD3 gamma CD3 deltaCD3 epsilon CD79a CD79b CD66d DAP10 DAP12

A primary intracellular signaling domain comprises a functionalfragment, or analog, of a primary stimulatory molecule (e.g., CD3zeta—GenBank Acc. No. BAG36664.1) It can comprise the entireintracellular region or a fragment of the intracellular region which issufficient for generation of an intracellular signal when an antigenbinding domain to which it is fused, or coupled by a dimerizationswitch, binds cognate antigen. In embodiments the primary intracellularsignaling domain has at least 70, 75. 80. 85, 90, 95, 98, or 99%sequence identity with a naturally occurring primary stimulatorymolecule, e.g., a human (GenBank Acc. No. BAG36664.1), or othermammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murineintracelluar primary stimulatory molecule. In embodiments the primaryintracellular signaling domain has at least 70, 75. 80. 85, 90, 95, 98,or 99% sequence identity with SEQ ID NO: 13.

In embodiments the primary intracellular signaling domain, has at least70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs byno more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residuesfrom the corresponding residues of a naturally occurring human primarystimulatory molecule, e.g., a naturally occurring human primarystimulatory molecule disclosed herein.

Costimulatory Signaling Domain

In an embodiment, a costimulatory signaling domain produces anintracellular signal when an extracellular domain, e.g., an antigenbinding domain to which it is fused, or coupled by a dimerizationswitch, binds cognate ligand. It is derived from a costimulatorymolecule. It comprises sufficient primary costimulatory moleculesequence to produce an intracellular signal, e.g., when an extracellulardomain, e.g., an antigen binding domain, to which it is fused, orcoupled by a dimerization switch, binds cognate ligand.

Costimulatory molecules are cell surface molecules, other than antigenreceptors or their counter ligands, that promote an immune effectorresponse. In some cases they are required for an efficient or enhancedimmune response. Typically, a costimulatory molecule generates anintracellular signal that is dependent on binding to a cognate ligandthat is, in embodiments, other than an antigen, e.g., the antigenrecognized by an antigen binding domain of a T cell. Typically,signaling from a primary stimulatory molecule and a costimulatorymolecule contribute to an immune effector response, and in some casesboth are required for efficient or enhanced generation of an immuneeffector response.

A costimulatory domain comprises a functional fragment, or analog, of acostimulatory molecule (e.g., 4-1BB). It can comprise the entireintracellular region or a fragment of the intracellular region which issufficient for generation of an intracellular signal, e.g., when anantigen binding domain to which it is fused, or coupled by adimerization switch, binds cognate antigen. In embodiments thecostimulatory domain has at least 70, 75. 80. 85, 90, 95, 98, or 99%sequence identity with a naturally occurring costimulatory molecule,e.g., a human, or other mammalian, e.g., a nonhuman species, e.g.,rodent, monkey, ape or murine intracelluar costimulatory molecule. Inembodiments the costimatory domain has at least 70, 75. 80. 85, 90, 95,98, or 99% sequence identity with SEQ ID NO: 12.

Exemplary costimulatory signaling domains (intracellular signalingdomains) are provided in Table 3.

TABLE 3 Costimulatory Signaling Domains for RCARX (identified by theCostimulatory Molecules from which they are derived) CD27 CD28, 4-1BB(CD137) OX40 CD30 CD40 ICOS (CD278) ICAM-1 LFA-1 (CD11a/CD18) CD2 CD7LIGHT NKG2C B7-H3 a ligand that specifically binds with CD83

In embodiments the costimulatory signaling domain, has at least 70, 75,80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no morethan 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from thecorresponding residues of a naturally occurring human primarystimulatory molecule, e.g., a naturally occurring human costimulatorymolecule disclosed herein.

Antigen Binding Domain

The CARs described herein, e.g., the KIR-CARs described herein, includean antigen binding domain in the extracellular region. An “antigenbinding domain” as the term is used herein, refers to a molecule thathas affinity for a target antigen, typically an antigen on a targetcell, e.g., a cancer cell. An exemplary antigen binding domain comprisesa polypeptide, e.g., an antibody molecule (which includes an antibody,and antigen binding fragments thereof, e.g., a immunoglobulin, singledomain antibody (sdAb), and an scFv), or a non-antibody scaffold, e.g.,a fibronectin, and the like. In embodiments, the antigen binding domainis a single polypeptide. In embodiments, the antigen binding domaincomprises, one, two, or more, polypeptides.

The choice of an antigen binding domain can depend upon the type andnumber of ligands or receptors that define the surface of a target cell.For example, the antigen binding domain may be chosen to recognize aligand or receptor that acts as a cell surface marker on target cellsassociated with a particular disease state. Examples of cell surfacemarkers that may act as ligands or receptors include a cell surfacemarker associated with a particular disease state, e.g., cell surfacemakers for viral diseases, bacterial diseases parasitic infections,autoimmune diseases and disorders associated with unwanted cellproliferation, e.g., a cancer, e.g., a cancer described herein.

In the context of the present disclosure, “tumor antigen” or“proliferative disorder antigen” or “antigen associated with aproliferative disorder” refers to antigens that are common to specificproliferative disorders. In certain aspects, the proliferative disorderantigens of the present invention are derived from, cancers includingbut not limited to primary or metastatic melanoma, thymoma, lymphoma,sarcoma, lung cancer (e.g., NSCLC or SCLC), liver cancer, non-Hodgkin'slymphoma, Hodgkin's lymphoma, leukemias, multiple myeloma, glioblastoma,neuroblastoma, uterine cancer, cervical cancer, renal cancer, thyroidcancer, bladder cancer, kidney cancer and adenocarcinomas such as breastcancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancerand the like. In some embodiments, the cancer is B-cell acute lymphoidleukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acutelymphoid leukemia (ALL), acute myelogenous leukemia (AML); one or morechronic leukemias including but not limited to chronic myelogenousleukemia (CML), chronic lymphocytic leukemia (CLL); additionalhematologic cancers or hematologic conditions including, but not limitedto B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cellneoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicularlymphoma, hairy cell leukemia, small cell- or a large cell-follicularlymphoma, malignant lymphoproliferative conditions, MALT lymphoma,mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma,myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia,

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes immunologically recognized by tumor infiltratinglymphocytes (TIL) derived from a cancer tumor of a mammal.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune responses. Theselection of the antigen binding domain of the invention will depend onthe particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, B7H3,Kit, CA-IX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionicgonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin B 1,lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2,RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1,fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerasereverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinalcarboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC,TRP-2, CYP1B1, BORIS, prostase, prostate-specific antigen (PSA), PAX3,PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p⁵³ mutant, Ras mutant, gp100,prostein, OR51E2, PANX3, PSMA, PSCA, Her2/neu, hTERT, HMWMAA, HAVCR1,VEGFR2, PDGFR-beta, legumain, HPV E6,E7, survivin and telomerase, spermprotein 17, SSEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumorantigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2,MelanA/MART1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17,neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1,ephrinB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171,CD179a, androgen receptor, insulin growth factor (IGF)-I, IGF-II, IGF-Ireceptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folatereceptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2,TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferredembodiment, the tumor antigen is selected from the group consisting offolate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33,BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof.

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-i, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target antigens includetransformation-related molecules such as the oncogene HER-2/Neu/ErbB-2.Yet another group of target antigens are onco-fetal antigens such ascarcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specificidiotype immunoglobulin constitutes a truly tumor-specificimmunoglobulin antigen that is unique to the individual tumor. B-celldifferentiation antigens such as CD19, CD20 and CD37 are othercandidates for target antigens in B-cell lymphoma.

Non-limiting examples of tumor antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp00 (Pmel 17),tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens suchas MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonicantigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen binddomain that is specific to the desired antigen target.

Antigen Binding Domains Derived from an Antibody Molecule

The antigen binding domain can be derived from an antibody molecule,e.g., one or more of monoclonal antibodies, polyclonal antibodies,recombinant antibodies, human antibodies, humanized antibodies,single-domain antibodies e.g., a heavy chain variable domain (VH), alight chain variable domain (VL) and a variable domain (VHH) from, e.g.,human or camelid origin. In some instances, it is beneficial for theantigen binding domain to be derived from the same species in which theCAR will ultimately be used in, e.g., for use in humans, it may bebeneficial for the antigen binding domain of the CAR, e.g., the KIR-CAR,e.g., described herein, to comprise a human or a humanized antigenbinding domain. Antibodies can be obtained using known techniques knownin the art.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with a target antigen. An antibody canbe intact immunoglobulin derived from natural sources or fromrecombinant sources and can be an immunoreactive portion of intactimmunoglobulin. Antibodies are typically tetramers of immunoglobulinmolecules. The antibody molecule described herein may exist in a varietyof forms where the antigen binding portion of the antibody is expressedas part of a contiguous polypeptide chain including, for example, asingle domain antibody fragment (sdAb), a single chain antibody (scFv)and a humanized or human antibody, e.g., as described herein.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, a single chain domain antibody (sdAb), Fab, Fab′, F(ab′)2, and Fvfragments, linear antibodies, scFv antibodies, a linear antibody, singledomain antibody such as an sdAb (either VL or VH), a camelid VHH domain,and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodymolecule which is generated using recombinant DNA technology, such as,for example, an antibody molecule expressed by a bacteriophage asdescribed herein. The term should also be construed to mean an antibodymolecule which has been generated by the synthesis of a DNA moleculeencoding the antibody molecule and which DNA molecule expresses anantibody protein, or an amino acid sequence specifying the antibody,wherein the DNA or amino acid sequence has been obtained using syntheticDNA or amino acid sequence technology which is available and well knownin the art.

In embodiments, the antigen binding domain comprises a fragment of anantibody that is sufficient to confer recognition and specific bindingto the target antigen. Examples of an antibody fragment include, but arenot limited to, an Fab, Fab′, F(ab′)₂, or Fv fragment, an scFv antibodyfragment, a linear antibody, single domain antibody such as an sdAb(either VL or VH), a camelid VHH domain, and multi-specific antibodiesformed from antibody fragments.

In an embodiment, the antigen binding domain is a “scFv,” which cancomprise a fusion protein comprising a VL chain and a VH chain of anantibody, where the VH and VL are, e.g., linked via a short flexiblepolypeptide linker, e.g., a linker described herein. The scFv is capableof being expressed as a single chain polypeptide and retains thespecificity of the intact antibody from which it is derived. Moreover,the VL and VH variable chains can be linked in either order, e.g., withrespect to the N-terminal and C-terminal ends of the polypeptide, thescFv may comprise VL-linker-VH or may comprise VH-linker-VL. An scFvthat can be prepared according to method known in the art (see, forexample, Bird et al., (1988) Science 242:423-426 and Huston et al.,(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

As described above and elsewhere, scFv molecules can be produced bylinking VH and VL chains together using flexible polypeptide linkers. Insome embodiments, the scFv molecules comprise flexible polypeptidelinker with an optimized length and/or amino acid composition. Theflexible polypeptide linker length can greatly affect how the variableregions of a scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids, intrachain foldingis prevented. For examples of linker orientation and size see, e.g.,Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S.Patent Application Publication Nos. 2005/0100543, 2005/0175606,2007/0014794, and PCT publication Nos. WO2006/020258 and WO2007/024715,is incorporated herein by reference. In one embodiment, the peptidelinker of the scFv consists of amino acids such as glycine and/or serineresidues used alone or in combination, to link variable heavy andvariable light chain regions together. In one embodiment, the flexiblepolypeptide linker is a Gly/Ser linker and, e.g., comprises the aminoacid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal toor greater than 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7,n=8, n=9 and n=10 (SEQ ID NO: 69). In one embodiment, the flexiblepolypeptide linkers include, but are not limited to, (Gly4 Ser)4 (SEQ IDNO: 70) or (Gly4 Ser)3 (SEQ ID NO: 71). In another embodiment, thelinkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser)(SEQ ID NO: 72).

In some embodiments, the antigen binding domain is a single domainantigen binding (SDAB) molecules. A SDAB molecule includes moleculeswhose complementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies (e.g., described in more detail below). SDAB molecules may beany of the art, or any future single domain molecules. SDAB moleculesmay be derived from any species including, but not limited to mouse,human, camel, llama, fish, shark, goat, rabbit, and bovine. This termalso includes naturally occurring single domain antibody molecules fromspecies other than Camelidae and sharks.

In one aspect, an SDAB molecule can be derived from a variable region ofthe immunoglobulin found in fish, such as, for example, that which isderived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainmolecules derived from a variable region of NAR (“IgNARs”) are describedin WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, an SDAB molecule is a naturally occurringsingle domain antigen binding molecule known as a heavy chain devoid oflight chains. Such single domain molecules are disclosed in WO 9404678and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.For clarity reasons, this variable domain derived from a heavy chainmolecule naturally devoid of light chain is known herein as a VHH ornanobody to distinguish it from the conventional VH of four chainimmunoglobulins. Such a VHH molecule can be derived from Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain moleculesnaturally devoid of light chain; such VHHs are within the scope of theinvention.

In certain embodiments, the SDAB molecule is a single chain fusionpolypeptide comprising one or more single domain molecules (e.g.,nanobodies), devoid of a complementary variable domain or animmunoglobulin constant, e.g., Fc, region, that binds to one or moretarget antigens.

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display).

In one embodiment, the antigen biding domain portion comprises a humanantibody or a fragment thereof.

In some embodiments, a non-human antibody is humanized, where specificsequences or regions of the antibody are modified to increase similarityto an antibody naturally produced in a human. In an embodiment, theantigen binding domain is humanized.

Non human antibodies can be humanized using a variety of techniquesknown in the art, e.g., CDR-grafting (see, e.g., European Patent No. EP239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos.5,225,539, 5,530,101, and 5,585,089, each of which is incorporatedherein in its entirety by reference), veneering or resurfacing (see,e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991,Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, ProteinEngineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973,each of which is incorporated herein by its entirety by reference),chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which isincorporated herein in its entirety by reference), and techniquesdisclosed in, e.g., U.S. Patent Application Publication No.US2005/0042664, U.S. Patent Application Publication No. US2005/0048617,U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, InternationalPublication No. WO 9317105, Tan et al., 2002, J. Immunol., 169:1119-25;Caldas et al., 2000, Protein Eng., 13(5):353-60; Morea et al., 2000,Methods, 20:267-79; Baca et al., 1997, J. Biol. Chem., 272:10678-84;Roguska et al., 1996, Protein Eng., 9(10):895-904; Couto et al., 1995,Cancer Res., 55:5973s-5977; Couto et al., 1995, Cancer Res.,55(8):1717-22; Sandhu 1994 Gene, 150(2):409-10; and Pedersen et al.,1994, J. Mol. Biol., 235(3):959-73, each of which is incorporated hereinin its entirety by reference. Often, framework residues in the frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, for example improve, antigen binding. Theseframework substitutions are identified by methods well-known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature, 332:323, which are incorporated hereinby reference in their entireties.). In preferred embodiments, thehumanized antibody molecule comprises a sequence described herein, e.g.,a variable light chain and/or a variable heavy chain described herein,e.g., a humanized variable light chain and/or variable heavy chaindescribed in Table 4.

A humanized antibody has one or more amino acid residues introduced intoit from a source which is nonhuman. These nonhuman amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Thus, humanized antibodies compriseone or more CDRs from nonhuman immunoglobulin molecules and frameworkregions from human. Humanization of antibodies is well-known in the artand can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized chimeric antibodies, substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some framework(FR) residues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies can also be achieved by veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology, 28(4/5):489-498; Studnicka et al., Protein Engineering,7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) orchain shuffling (U.S. Pat. No. 5,565,332), the contents of which areincorporated herein by reference herein in their entirety.

In some embodiments, the antibody of the invention is further preparedusing an antibody having one or more of the VH and/or VL sequencesdisclosed herein can be used as starting material to engineer a modifiedantibody, which modified antibody may have altered properties ascompared to the starting antibody. In various embodiments, the antibodyis engineered by modifying one or more amino acids within one or bothvariable regions (i.e., VH and/or VL), for example within one or moreCDR regions and/or within one or more framework regions.

In another aspect, the antigen binding domain is a T cell receptor(“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR).Methods to make such TCRs is known in the art. See, e.g., Willemsen R Aet al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012)(references are incorporated herein by its entirety). For example, scTCRcan be engineered that contains the Vα and Vβ genes from a T cell clonelinked by a linker (e.g., a flexible peptide). This approach is veryuseful to cancer associated target that itself is intracellular,however, a fragment of such antigen (peptide) is presented on thesurface of the cancer cells by MHC.

TABLE 4 Exemplary Antigen Binding Domains Target Antigen NameAmino Acid Sequence CD19 huscFv1EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 16) CD19 huscFv2eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 17) CD19 huscFv3qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 18) CD19 huscFv4qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 19) CD19 huscFv5eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 20) CD19 huscFv6eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 21) CD19 huscFv7qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 22) CD19huscFv8qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 23) CD19huscFv9eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 24) CD19 HuqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiscFv10 skdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 25) CD19 HueivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdyscFv11tltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 26) CD19 HuqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiscFv12 skdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 27) CD19 muCTLdiqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdy019sltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvss (SEQ ID NO: 28) CD123 Mu 1172DIVLTQSPASLAVSLGQRATISCRASESVDNYGNTFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPPTFGAGTKLELKGGGGSGGGGSSGGGSQIQLVQSGPELKKPGETVKISCKASGYIFTNYGMNWVKQAPGKSFKWMGWINTYTGESTYSADFKGRFAFSLETSASTAYLHINDLKNEDTATYFCARSGGYDPMDYWGQGTSVTVSS (SEQ ID NO: 29) CD123 Mu 1176DVQITQSPSYLAASPGETITINCRASKSISKDLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNKYPYTFGGGTKLEIKGGGGSGGGGSSGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPDQGLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYW GQGTTLTVSS (SEQ ID NO: 30)CD123 huscFv1divltqspdslayslgeratincrasesvdnygntfmhwyqqkpgqppklliyrasnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsnedpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgselkkpgasvkvsckasgyiftnygmnwvrqapgqglewmgwintytgestysadfkgrfvfsldtsystaylqinalkaedtavyycarsggydpmdywgqgttvtvss (SEQ ID NO: 31) CD123huscFv2divltqspdslayslgeratincrasesvdnygntfmhwyqqkpgqppklliyrasnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsnedpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgaevkkpgasvkvsckasgyiftnygmnwvrqapgqrlewmgwintytgestysadfkgrvtitldtsastaymelsslrsedtavyycarsggydpmdywgqgttvtvss (SEQ ID NO: 32) CD123huscFv3eivltqspatlslspgeratlscrasesvdnygntfmhwyqqkpgqaprlliyrasnlesgiparfsgsgsrtdftltisslepedvavyycqqsnedpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgselkkpgasvkvsckasgyiftnygmnwvrqapgqglewmgwintytgestysadfkgrfvfsldtsystaylqinalkaedtavyycarsggydpmdywgqgttvtvss (SEQ ID NO: 33) CD123huscFv4eivltqspatlslspgeratlscrasesvdnygntfmhwyqqkpgqaprlliyrasnlesgiparfsgsgsrtdftltisslepedvavyycqqsnedpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgaevkkpgasvkvsckasgyiftnygmnwvrqapgqrlewmgwintytgestysadfkgrvtitldtsastaymelsslrsedtavyycarsggydpmdywgqgttvtvss (SEQ ID NO: 34) CD123huscFv5qiqlvqsgselkkpgasvkvsckasgyiftnygmnwvrqapgqglewmgwintytgestysadfkgrfvfsldtsystaylqinalkaedtavyycarsggydpmdywgqgttvtvssggggsggggsggggsggggsdivltqspdslayslgeratincrasesvdnygntfmhwyqqkpgqppklliyrasnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsnedpptfgqgtkleik (SEQ ID NO: 35) CD123huscFv6qiqlvqsgselkkpgasvkvsckasgyiftnygmnwvrqapgqglewmgwintytgestysadfkgrfvfsldtsystaylqinalkaedtavyycarsggydpmdywgqgttvtvssggggsggggsggggsggggseivltqspatlslspgeratlscrasesvdnygntfmhwyqqkpgqaprlliyrasnlesgiparfsgsgsrtdftltisslepedvavyycqqsnedpptfgqgtkleik (SEQ ID NO: 36) CD123huscFv7qiqlvqsgaevkkpgasvkvsckasgyiftnygmnwvrqapgqrlewmgwintytgestysadfkgrvtitldtsastaymelsslrsedtavyycarsggydpmdywgqgttvtvs sggggsggggsggggsggggsdivltqspdslayslgeratincrasesvdnygntfmhwyqqkpgqppklliyrasnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsnedpptfgqgtkleik (SEQ ID NO: 37) CD123huscFv8qiqlvqsgaevkkpgasvkvsckasgyiftnygmnwvrqapgqrlewmgwintytgestysadfkgrvtitldtsastaymelsslrsedtavyycarsggydpmdywgqgttvtvssggggsggggsggggsggggseivltqspatlslspgeratlscrasesvdnygntfmhwyqqkpgqaprlliyrasnlesgiparfsgsgsrtdftltisslepedvavyycqqsnedpptfgqgtkleik (SEQ ID NO: 38) EGFRhuscFv1eiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifq vIIIgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqspdslayslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveik (SEQ ID NO: 39) EGFRhuscFv2dvvmtqspdslayslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgvIIIsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvss (SEQ ID NO: 40) EGFR huscFv3eiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifq vIIIghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveik (SEQ ID NO: 41) EGFRhuscFv4dvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsvIII gsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvss (SEQ ID NO: 42) EGFRhuscFv5eiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifq VIIIgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveik (SEQ ID NO: 43) EGFR huscFv6eiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifq vIIIghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqspdslayslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveik (SEQ ID NO: 44) EGFRhuscFv7dvvmtqspdslayslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgVIIIsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvss (SEQ ID NO: 45) EGFRhuscFv8dvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsvIII gsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvss (SEQ ID NO: 46) EGFR Mu310CeiqlqqsgaelvkpgasvklsctgsgfniedyyihwvkqrteqglewigridpendetkygpifqgravIIItitadtssntvylqlssltsedtavyycafrggvywgpgttltvssggggsggggsggggshmdvvmtqspltlsvaigqsasisckssqslldsdgktylnwllqrpgqspkrlislvskldsgvpdrftgsgsgtdftlrisrveaedlgiyycwqgthfpgtfgggtkleik (SEQ ID NO: 47) meso- ss1QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQS thelinHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEI (SEQ ID NO: 48)

Non-Antibody Scaffolds

In embodiments, the antigen binding domain comprises a non antibodyscaffold, e.g., a fibronectin, ankyrin, domain antibody, lipocalin,small modular immuno-pharmaceutical, maxybody, Protein A, or affilin.The non antibody scaffold has the ability to bind to target antigen on acell. In embodiments, the antigen binding domain is a polypeptide orfragment thereof of a naturally occurring protein expressed on a cell.In some embodiments, the antigen binding domain comprises a non-antibodyscaffold. A wide variety of non-antibody scaffolds can be employed solong as the resulting polypeptide includes at least one binding regionwhich specifically binds to the target antigen on a target cell.

Non-antibody scaffolds include: fibronectin (Novartis, Mass.), ankyrin(Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil ProteinsGmbH, Halle, Germany).

Fibronectin scaffolds can be based on fibronectin type III domain (e.g.,the tenth module of the fibronectin type III (¹⁰Fn3 domain)). Thefibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). Because of this structure, this non-antibody scaffold mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat module is a 33amino acid polypeptide consisting of two anti-parallel t-helices and aP3-turn. Binding of the variable regions is mostly optimized by usingribosome display.

Avimers are derived from natural A-domain containing protein such asHER3. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, U.S.Patent Application Publication Nos. 20040175756; 20050053973;20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-likemolecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures ofproteins, the major secondary structure involved in protein-proteininteractions

Mismatched Antigen Binding Domains

It has been discovered, that cells having a plurality of chimericmembrane embedded receptors each comprising an antigen binding domain(CMERs) that interactions between the antigen binding domain of the CMERcan be undesirable, e.g., because it inhibits the ability of one or moreof the antigen binding domains to bind its cognate antigen. Accordingly,disclosed herein are a first and a second non-naturally occurring CMERcomprising antigen binding domains that minimize such interactions whenexpressed in the same cell. In an embodiment a plurality of CMERscomprises two TCARs. In an embodiment a plurality of CMERs comprises aTCAR and another CMER. In an embodiment a plurality of CMERs comprisestwo NKR-CARs. In an embodiment a plurality of CMERs comprises a NKR-CARand another CMER. In an embodiment a plurality of CMERs comprises a TCARand an NKR-CAR.

In some embodiments, the claimed invention comprises a first and secondCMER, wherein the antigen binding domain of one of said first CMER saidsecond CMER does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofsaid first CMER said second CMER is an scFv, and the other is not anscFv. In some embodiments, the antigen binding domain of one of saidfirst CMER said second CMER comprises a single VH domain, e.g., acamelid, shark, or lamprey single VH domain, or a single VH domainderived from a human or mouse sequence or a non-antibody scaffold. Insome embodiments, the antigen binding domain of one of said first CMERsaid second CMER comprises a nanobody. In some embodiments, the antigenbinding domain of one of said first CMER said second CMER comprises acamelid VHH domain.

In some embodiments, the antigen binding domain of one of said firstCMER said second CMER comprises an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence. In someembodiments, the antigen binding domain of one of said first CMER saidsecond CMER comprises an scFv, and the other comprises a nanobody. Insome embodiments, the antigen binding domain of one of said first CMERsaid second CMER comprises an scFv, and the other comprises a camelidVHH domain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of said first CMER to its cognate antigen isnot substantially reduced by the presence of said second CMER. In someembodiments, binding of the antigen binding domain of said first CMER toits cognate antigen in the presence of said second CMER is 85%, 90%,95%, 96%, 97%, 98% or 99% of binding of the antigen binding domain ofsaid first CMER to its cognate antigen in the absence of said secondCMER.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of said first CMER said second CMER, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of said first CMER said secondCMER, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99%less than if both were scFv antigen binding domains.

In some embodiments, the claimed invention comprises a first and secondKIR-CAR, wherein the antigen binding domain of one of said first KIR-CARsaid second KIR-CAR does not comprise a variable light domain and avariable heavy domain. In some embodiments, the antigen binding domainof one of said first KIR-CAR said second KIR-CAR is an scFv, and theother is not an scFv. In some embodiments, the antigen binding domain ofone of said first KIR-CAR said second KIR-CAR comprises a single VHdomain, e.g., a camelid, shark, or lamprey single VH domain, or a singleVH domain derived from a human or mouse sequence or a non-antibodyscaffold. In some embodiments, the antigen binding domain of one of saidfirst KIR-CAR said second KIR-CAR comprises a nanobody. In someembodiments, the antigen binding domain of one of said first KIR-CARsaid second KIR-CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of said firstKIR-CAR said second KIR-CAR comprises an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence. In someembodiments, the antigen binding domain of one of said first KIR-CARsaid second KIR-CAR comprises an scFv, and the other comprises ananobody. In some embodiments, the antigen binding domain of one of saidfirst KIR-CAR said second KIR-CAR comprises an scFv, and the othercomprises a camelid VHH domain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of said first KIR-CAR to its cognate antigenis not substantially reduced by the presence of said second KIR-CAR. Insome embodiments, binding of the antigen binding domain of said firstKIR-CAR to its cognate antigen in the presence of said second KIR-CAR is85%, 90%, 95%, 96%, 97%, 98% or 99% of binding of the antigen bindingdomain of said first KIR-CAR to its cognate antigen in the absence ofsaid second KIR-CAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of said first KIR-CAR said second KIR-CAR, associatewith one another less than if both were scFv antigen binding domains. Insome embodiments, the antigen binding domains of said first KIR-CAR saidsecond KIR-CAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98%or 99% less than if both were scFv antigen binding domains.

In some embodiments, the claimed invention comprises a first and secondTCAR, wherein the antigen binding domain of one of said first TCAR saidsecond TCAR does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofsaid first TCAR said second TCAR is an scFv, and the other is not anscFv. In some embodiments, the antigen binding domain of one of saidfirst TCAR said second TCAR comprises a single VH domain, e.g., acamelid, shark, or lamprey single VH domain, or a single VH domainderived from a human or mouse sequence or a non-antibody scaffold. Insome embodiments, the antigen binding domain of one of said first TCARsaid second TCAR comprises a nanobody. In some embodiments, the antigenbinding domain of one of said first TCAR said second TCAR comprises acamelid VHH domain.

In some embodiments, the antigen binding domain of one of said firstTCAR said second TCAR comprises an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence. In someembodiments, the antigen binding domain of one of said first TCAR saidsecond TCAR comprises an scFv, and the other comprises a nanobody. Insome embodiments, the antigen binding domain of one of said first TCARsaid second TCAR comprises an scFv, and the other comprises a camelidVHH domain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of said first TCAR to its cognate antigen isnot substantially reduced by the presence of said second TCAR. In someembodiments, binding of the antigen binding domain of said first TCAR toits cognate antigen in the presence of said second TCAR is 85%, 90%,95%, 96%, 97%, 98% or 99% of binding of the antigen binding domain ofsaid first TCAR to its cognate antigen in the absence of said secondTCAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of said first TCAR said second TCAR, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of said first TCAR said secondTCAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99%less than if both were scFv antigen binding domains.

Vectors

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. In brief summary, the expression of natural or syntheticnucleic acids encoding CARs is typically achieved by operably linking anucleic acid encoding the CAR polypeptide or portions thereof to apromoter, and incorporating the construct into an expression vector. Thevectors can be suitable for replication and integration eukaryotes.Typical cloning vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

An example of a promoter is the immediate early cytomegalovirus (CMV)promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the elongation factor-1α promoter, as well as human genepromoters such as, but not limited to, the actin promoter, the myosinpromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In one embodiment, the vector is a lentivirus vector. In one embodiment,the vector further comprises a promoter. In one embodiment, the promoteris an EF-1 promoter.

In one embodiment, the vector is an in vitro transcribed vector, e.g., avector that transcribes RNA of a nucleic acid molecule described herein.In one embodiment, the nucleic acid sequence in the vector furthercomprises a poly(A) tail, e.g., a poly A tail described herein, e.g.,comprising about 150 adenosine bases. In one embodiment, the nucleicacid sequence in the vector further comprises a 3′UTR.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Polynucleotides can be introduced into target cells using any of anumber of different methods, for instance, commercially availablemethods which include, but are not limited to, electroporation (AmaxaNucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX)(Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad,Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationicliposome mediated transfection using lipofection, polymer encapsulation,peptide mediated transfection, or biolistic particle delivery systemssuch as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

Disclosed herein are methods for producing an in vitro transcribed RNANK-CAR. The present invention also includes an NK-CAR encoding RNAconstruct that can be directly transfected into a cell. A method forgenerating mRNA for use in transfection can involve in vitrotranscription (IVT) of a template with specially designed primers,followed by polyA addition, to produce a construct containing 3′ and 5′untranslated sequence (“UTR”), a 5′ cap and/or Internal Ribosome EntrySite (IRES), the nucleic acid to be expressed, and a polyA tail,typically 50-2000 bases in. RNA so produced can efficiently transfectdifferent kinds of cells. In one aspect, the template includes sequencesfor the NK-CAR.

In one aspect the NK-CAR is encoded by a messenger RNA (mRNA). In oneaspect the mRNA encoding the NK-CAR is introduced into a T cell forproduction of a NK-CAR cell.

In one embodiment, the in vitro transcribed RNA NK-CAR can be introducedto a cell as a form of transient transfection. The RNA is produced by invitro transcription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. In one embodiment, the desired templefor in vitro transcription is a NK-CAR of the present invention. Forexample, the template for the RNA NK-CAR comprises an extracellularregion comprising a single chain variable domain of an anti-tumorantibody; a hinge region, a transmembrane domain (e.g., a transmembranedomain of KIR). In one embodiment, the desired temples for in vitrotranscription comprises KIR-CAR and DAP12 on separate templates. In oneembodiment, the desired temple for in vitro transcription comprisesKIR-CAR and DAP12 on the same template. The template for DAP12 comprisesa transmembrane domain and an intracellular region.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the nucleic acid can includesome or all of the 5′ and/or 3′ untranslated regions (UTRs). The nucleicacid can include exons and introns. In one embodiment, the DNA to beused for PCR is a human nucleic acid sequence. In another embodiment,the DNA to be used for PCR is a human nucleic acid sequence includingthe 5′ and 3′ UTRs. The DNA can alternatively be an artificial DNAsequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary,” as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5′ and 3′ UTRs.Primers useful for PCR can be generated by synthetic methods that arewell known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. Alternatively, UTR sequences thatare not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3′ UTR sequences candecrease the stability of mRNA. Therefore, 3′ UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5′ UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5′ UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3′or 5′ UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100 T tail (SEQ ID NO: 73) (size can be 50-5000 T (SEQ ID NO: 74)), orafter PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Generally, the length of a poly(A)tail positively correlates with the stability of the transcribed RNA. Inone embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQID NO: 75). Poly(A) tails of RNAs can be further extended following invitro transcription with the use of a poly(A) polymerase, such as E.coli polyA polymerase (E-PAP). In one embodiment, increasing the lengthof a poly(A) tail from 100 nucleotides (SEQ ID NO: 76) to between 300and 400 nucleotides (SEQ ID NO: 77) results in about a two-fold increasein the translation efficiency of the RNA. Additionally, the attachmentof different chemical groups to the 3′ end can increase mRNA stability.Such attachment can contain modified/artificial nucleotides, aptamersand other compounds. For example, ATP analogs can be incorporated intothe poly(A) tail using poly(A) polymerase. ATP analogs can furtherincrease the stability of the RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded. In the case where a non-viral delivery system is utilized, anexemplary delivery vehicle is a liposome. The use of lipid formulationsis contemplated for the introduction of the nucleic acids into a hostcell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acidmay be associated with a lipid. The nucleic acid associated with a lipidmay be encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Sources of T Cells

Prior to expansion and genetic modification, a source of T cells isobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable in the art, may be used. In certain embodiments of the presentinvention, T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred embodiment, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one embodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutionwith or without buffer. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly resuspended inculture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 10 per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

T cells are activated and expanded generally using methods as described,for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680;6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318;7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514;6,867,041; and U.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besançon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle:cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain embodiments, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one embodiment, a concentrationof about 2 billion cells/ml is used. In another embodiment, greater than100 million cells/ml is used. In a further embodiment, a concentrationof cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml isused. In yet another embodiment, a concentration of cells from 75, 80,85, 90, 95, or 100 million cells/ml is used. In further embodiments,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainembodiments. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) modified toexpress a plurality of types of KIR-CARs, wherein each KIR-CAR combinesan antigen recognition domain of a specific antibody with a component ofa KIR.

In one embodiment, the KIR-CARs of the invention comprise an activatingKIR which delivers its signal through an interaction with theimmunotyrosine-based activation motif (ITAM) containing membraneprotein, DAP12 that is mediated by residues within the transmembranedomains of these proteins.

In one embodiment, the KIR-CARs of the invention comprise an inhibitoryKIR which delivers its signal through one or more immunotyrosine-basedinhibitory motifs (ITIMs) that interact directly or indirectly withcytoplasmic signaling proteins such as SHP-1, SHP-2 and Vav family ofproteins. KIRs bearing cytoplasmic domains that contain (ITIMs) abrogatethe activating signal leading to inhibition of NK cytolytic and cytokineproducing activity. In some instances, the modified T cell expressing aKIR-CAR of the invention can elicit a KIR-CAR-mediated T-cell response.In one embodiment, the dependence of the binding to more than one typeof antigen allows the modified T cell to exhibit a heightenedspecificity to elicit a response upon binding of a tumor cell ratherthan a normal bystander cell.

The invention provides the use of a plurality of types of KIR-CARs toredirect the specificity of a primary T cell to a tumor antigen. Thus,the present invention also provides a method for stimulating a Tcell-mediated immune response to a target cell population or tissue in amammal comprising the step of administering to the mammal a T cell thatexpresses a plurality of types of KIR-CARs, wherein each type of KIR-CARcomprises a binding moiety that specifically interacts with apredetermined target. In one embodiment, the cell comprises a firstKIR-CAR comprising an activating KIR (actKIR-CAR), and a second KIR-CARcomprising an inhibitory KIR (inhKIR-CAR).

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the KIR-CAR-modified T cells may be anactive or a passive immune response. In addition, the KIR-CAR mediatedimmune response may be part of an adoptive immunotherapy approach inwhich KIR-CAR-modified T cells induce an immune response specific to theantigen binding domain in the KIR-CAR.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the antigen bind moiety portion of the KIR-CAR Tcells of the invention is designed to treat a particular cancer. In oneembodiment, the KIR-CAR T cells of the invention are modified to expressa first actKIR-CAR targeting a first antigen and a second inhKIR-CARtargeting a second antigen, where the first antigen is expressed on aparticular tumor or cancer and the second antigen is not expressed on aparticular tumor or cancer. In this manner, conditional activation of Tcells is generated by engagement of actKIR-CAR (or standard TCR-zeta CARbearing a scFv to an antigen on the malignant cell of interest) and theinhKIR-CAR bearing for example a scFv directed against an antigen thatis present on normal, but not malignant tissue provides inhibition ofthe activating signal from the actKIR-CAR when the KIR-CAR T cellencounters normal cells. Examples of antigens that serve as usefultargets for inhibitory CARs include the ephrin receptors (Pasquale,2010, Nat Rev Cancer 10(3):165-80) and claudins (Singh et al., 2010, JOncol, 2010:541957), which are expressed by epithelial cells from normaltissues, but often selectively lost by cancers (e.g. EPHA7).

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding aKIR-CAR to the cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a KIR-CAR disclosed herein. TheKIR-CAR-modified cell can be administered to a mammalian recipient toprovide a therapeutic benefit. The mammalian recipient may be a humanand the KIR-CAR-modified cell can be autologous with respect to therecipient. Alternatively, the cells can be allogeneic, syngeneic orxenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, theKIR-CAR-modified T cells of the invention are used in the treatment ofcancer. In certain embodiments, the cells of the invention are used inthe treatment of patients at risk for developing cancer. Thus, thepresent invention provides methods for the treatment or prevention ofcancer comprising administering to a subject in need thereof, atherapeutically effective amount of the KIR-CAR-modified T cells of theinvention.

The KIR-CAR-modified T cells of the present invention may beadministered either alone, or as a pharmaceutical composition incombination with diluents and/or with other components such as IL-2 orother cytokines or cell populations. Briefly, pharmaceuticalcompositions of the present invention may comprise a target cellpopulation as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arepreferably formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to draw blood (or have anapheresis performed), activate and genetically modify the T cellstherefrom according to the present invention, and reinfuse the patientwith these activated and expanded genetically modified T cells. Thisprocess can be carried out multiple times every few weeks. In certainembodiments, T cells can be activated from blood draws of from 10 cc to400 cc. In certain embodiments, T cells are activated from blood drawsof 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.Not to be bound by theory, using this multiple blood draw/multiplereinfusion protocol may serve to select out certain populations of Tcells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices.Strategies for CAR T cell dosing and scheduling have been discussed(Ertl et al, 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal ofTranslational Medicine, 8:55).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Chimeric NK Receptors

The results presented herein demonstrate an alternative approach toconstructing CARs for T cells that can be more finely regulated comparedwith current CAR designs. Experiments were designed to develop a novel,regulated CAR system that comprises at least two or three chimericfusion proteins. The primary T cell activating signal and inhibitorysignals are based upon naturally occurring activating and inhibitoryreceptors of NK cells known as killer cell immunoglobulin-like receptors(KIRs).

KIRs exist as both activating and inhibitory forms that depend upon theintracellular domain of the receptor. Activating KIRs deliver theirsignals through an interaction with the immunotyrosine-based activationmotif (ITAM) containing membrane protein, DAP12 that is recruited byresidues within the transmembrane domains of these proteins. InhibitoryKIRs bear a cytoplasmic domain that contains immunotyrosine-basedinhibitory motifs (ITIMs), which abrogate the activating signal leadingto inhibition of NK cytolytic and cytokine producing activity. Similarto TCRs, KIRs belong to the immunoglobulin family of protein receptors,and many bind to invariant MHC and MHC-like ligands. Without wishing tobe bound by any particular theory, it is believed that theseinteractions are utilized to naturally distinguish normal cells (usuallyexpressing high density MHC class I) from malignant or virally infectedcells (often with low or missing MHC class I).

KIR-like chimeric antigen receptors (KIR-CARs) have been constructedwhich fuse an scfv to a target antigen of interest with activating andinhibitory KIRs as shown in FIGS. 1A and 1B. Conditional activation of Tcells is generated by engagement of an activating KIR-CAR (actKIR-CAR)or standard TCR-zeta CAR bearing an scfv to an antigen on the malignantcell of interest. An inhibitory CAR (inhCAR) bearing an scfv directedagainst an antigen that is present on normal, but not malignant tissuewould provide dampening of the activating CAR primary signal when the Tcell encounters normal cells. Examples of antigens that serve as usefultargets for inhibitory CARs include the ephrin receptors (Pasquale,2010, Nat Rev Cancer 10(3):165-80) and claudins (Singh et al., 2010, JOncol, 2010:541957), which are expressed by epithelial cells from normaltissues, but often selectively lost by cancers (e.g. EPHA7).

Example 2: Activating KIR-CAR Construction and Activity

Experiments were designed to construct activating KIR-CARs based uponfusion of the anti-CD19 or anti-mesothelin scFv (SS-1) that werepreviously incorporated into CARs based upon the TCR-zeta cytoplasmicdomain that are currently in clinical trials. The human KIR2DS2activating KIR receptor was chosen as the initial base receptor for theactKIR-CAR. In order to deliver activating signals, the actKIR-CARsrequired coexpression of DAP12, which is not expressed normally in Tcells. Therefore, a lentiviral vector that expresses both the actKIR-CARwith human DAP12 using a “bicistronic” gene cassette based upon the 2Aribosomal skip peptide was constructed. A diagram of the lentiviralvector is illustrated in FIG. 2. Initial studies demonstrated that theactKIR-CARs were efficiently expressed in primary human T cells and theSS1 actKIR-CAR bound to mesothelin (FIG. 3). Similar to the previouslydeveloped and published SS1 scFv CD3 zeta (SS1-ζ) CAR (Carpenito et al.,2009, Proc Natl Acad Sci USA 106(9):3360-5), T cells expressing the SS1actKIR-CAR demonstrated cytotoxic activity towards target K562 cellsengineered to express the mesothelin ligand (KT-meso) as shown in FIG.4. Neither receptor exhibits killing of wild-type K562 lacking themesothelin target.

Since the cytotoxic activity of the SS1 KIR CAR towardsmesothelin-positive target cells was lower than the standardTCRzeta-based CAR targeting the same antigen with comparable CAR surfaceexpression, it is believed that the mesothelin CAR may have anextracellular hinge (based upon wild-type KIR2DS2) that is non-optimalfor segregation from CD45 due to its length. The kinetic segregation ofactivating ITAM-based receptors from CD45 is believed to be a keymechanisms for TCR activation, and dependent upon a length scale betweenthe T cell and target cell membranes of ˜14-15 nm (Choudhuri et al.,2005, Nature 436(7050):578-82). It is estimated that the KIR2DS2 basedSS 1 KIR-CAR to have a length scale of greater than 20 nm based upon thepartial crystal structure of mesothelin demonstrating that the SS1epitope is likely at an ˜10 nm distance from the target cell membrane(Ma et al., 2012, J Biol Chem 287(40):33123-31) and CAR that isestimated to be ˜10 nm assuming each Ig-like domain is ˜3.5 nm in theKIR2DS2 hinge in addition to the scFv. Therefore an activating KIR CARin which the KIR2DS2 hinge was removed (KIRS2 CAR) as shownschematically in FIGS. 5A and 5B was constructed. It was shown that anSS1 scFv based KIRS2 CAR exhibited enhanced cytolytic activity towardsmesothelin-expressing target cells compared with the CAR formed byfusion of the SS1 scFv onto full length wildtype KIR2DS2 (FIGS. 6A and6B). This optimized KIRS2 CAR also showed enhanced activity over the SS1scFv based TCRzeta CAR having a CD8 alpha extracellular hinge.

Example 3: InhKIR-CAR Construction and Activity

An inhibitory KIR-CAR was constructed based upon the fusion of theanti-mesothelin SS 1 scFv to the inhibitory KIR2DL3 receptor base.Initial studies demonstrated that the inhKIR-CARs efficiently expressedin primary human T cells. CD19 actKIR-CAR, SS1 actKIR-CAR and SS1inhKIR-CAR alone or in combination have been introduced into Jurkat Tcells bearing a dsGFP reporter under the control of an NFAT-drivenpromoter to monitor activation of this critical T cell signalingpathway. While Jurkat T cells expressing CD19 actKIR-CAR or SS1actKIR-CAR alone are efficiently activated by K562 expressing both CD19and mesothelin (KT-meso/CD19), Jurkat T cells co-expressing the CD19actKIR-CAR and the SS1 inhKIR-CAR showed markedly reduced activation bythe same KT-meso/CD19 target cells (FIG. 7A); however, analysis of thesurface expression of the CD19 and mesothelin scFv binding usingidiotype specific reagents surprisingly demonstrated that the expressionof the different scFv target specificities were mutually exclusive (FIG.7B).

Example 4: Sensitivity of Activating KIR-CAR Designs to NaturalInhibitory Receptor Systems

Since co-expression of two scFv CARs is limited, a strategy was pursuedto evaluate the sensitivity of the KIR-based activating CARs toinhibitory signals derived from the PD-1 receptor. PD-1 is a naturalreceptor in T cells that uses an ITIM in the cytoplasmic domain similarto inhibitory KIRs to recruit phosphatases that negatively regulate TCRsignaling. A schematic representation is shown in FIG. 19. The resultspresented herein demonstrate that wild-type PD-1 can be over-expressedwith both an activating KIR-based CAR and a TCR-zeta based CAR targetingmesothelin (FIGS. 8A and 8C). The results also show that thiscombination led to PD-1 ligand 1 (PDL-1) dependent inhibition of themesothelin-specific activating KIR-CAR cytotoxicity (FIG. 9). In thecontext of normal PD-1 expression by the T cells (i.e. T cells withoutPD-1 transfection), the KIR-CAR exhibits less inhibition whenencountering PD-L1 overexpressing target cells compared with theTCR-zeta based CAR. Without wishing to be bound by any particulartheory, it is believed that this may be an advantage of the KIR-CARswhen encountering tumors that commonly express inhibitory receptorligands.

Example 5: Co-Stimulation Dependent Activation of KIR CARs

Experiments were designed to evaluate the effects of chimericco-stimulatory receptors (CCRs) in the KIR-CAR system compared to thatdescribed with standard CARs by Kloss et al. (Kloss et al., 2013, NatBiotechnol 31(1):71-5). Experiments have also been designed to evaluatethe costimulatory dependent activation requirements for KIRs by engagingthe endogenous CD28 receptor in T cell using the agonist antibody, clone9.3. As shown in FIG. 14, the KIRS2 CAR showed robust proliferation inresponse to mesothelin-positive targets in the absence of CD28costimulation. This proliferation is superior to that observed with aTCR-zeta CAR where co-stimulation has been shown to be critical toproliferation. This data suggests that the KIR-based CAR may not havethe same costimulation requirements as TCR-zeta CARs forantigen-specific proliferation (Milone et al., 2009, Mol Ther17(8):1453-64; Carpenito et al., 2009, Proc Natl Acad Sci USA106(9):3360-5), and this costimulation independence may be a significantadvantage of KIR-based CARs to current TCR-zeta-based CARs. Experimentshave been designed to evaluate the KIR-based CARs in humanized mice totest the KIR-based CAR against CARs with and without costimulatorydomains in an in vivo pre-clinical setting (data and experiments inexample 5 are also presented in example 6).

Example 6: Killer Immunoglobulin-Like Receptor (KIR)-Based ChimericAntigen Receptors (CARs) Trigger Robust Cytotoxic Activity in SolidTumors

Chimeric antigen receptors (CARs) bearing an antigen-binding domainlinked in cis to the cytoplasmic domains of CD3- and costimulatoryreceptors provide a potent method for engineering T cell cytotoxicitytowards tumors (Grupp et al., The New England journal of medicine,368(16): 1509-18, 2013; Brentjens et al., Science translationalmedicine, 5(177):177ra38, 2013; Porter et al., The New England journalof medicine, 365(8):725-33, 2011). An alternative chimeric receptor inwhich a single chain variable fragment (scFv) targeting mesothelin (SS1)was fused to the transmembrane and cytoplasmic domain of KIR2DS2, astimulatory killer immunoglobulin-like receptor (KIR) normally expressedby natural killer (NK) cells is described herein. This SS1-KIRS2KIR-based CAR triggers robust antigen-specific cytotoxic activity,cytokine secretion and proliferation when delivered to T cells incombination with adaptor molecule DAP12. Importantly, using a xenograftmodel of mesothelioma that is resistant to CD3ζ-based CAR-modified Tcells bearing the cytoplasmic domains from the costimulatory receptors,CD28 or 4-1BB, the SS1-KIRS2/DAP12-modified T cells exhibit superioranti-tumor activity, suggesting that the KIR-based CAR can overcomeinhibitory signals within tumors that limit second and third generationCD3ζ-based CARs. The data presented herein support future clinicalevaluation of a KIR-based CAR in solid tumors.

“First generation” CARs were designed by the incorporation of acytoplasmic domain containing the immunotyrosine-based activation motif(ITAM) into a single chimeric receptor that uses a single chain variablefragment (scFv) from an antibody for specific antigen targeting(Sadelain et al., Cancer discovery, 3(4):388-98, 2013). A number ofdifferent additional signaling domains from co-stimulatory receptorssuch as CD28, ICOS, 4-1BB and OX-40 were later incorporated in tandeminto these receptors to enhance the proliferation and effector functionof CARs (Finney H M et al. J Immunol. 1998; 161:2791-2797; Maher J. etal. Nat Biotech 2002; 20:70-75; Finney H M et al. J Immunol. 2004;18:676-684; Milone et al., 2009, Mol Ther 17(8):1453-64; Carpenito etal., 2009, Proc Natl Acad Sci USA 106(9):3360-5). These “secondgeneration” (one co-stimulatory domain) and “third generation” (2co-stimulatory domains) CARs demonstrate significantly enhanced functionin preclinical animal models of cancer, and theseco-stimulation-enhanced CARs are currently in human clinical trials forhematologic malignancies and solid tumors (reviewed in Barrett D M etal. Ann Rev Med 2014; 65:333-347).

Although single-chain CARs trigger robust antigen-specific cytotoxicactivity, natural receptors utilizing the highly conserved ITAM domainsare generally structured into multi-chain complexes composed of separateligand binding and ITAM-containing signaling chains, such as the T cellreceptor (TCR)-CD3 complex, the B cell receptor (BCR)-Iga/P complex andthe Fc receptor (FcR) complex. The benefits of a multi-chainimmunoreceptor complex have been postulated to include: 1) greaterdiversity of signals available through the multiple chain interactions,2) the use of one signaling domain for multiple ligand-bindingreceptors, and 3) sustained signaling by the ITAM-containing chain thatis separable from the internalization of the ligand-binding chain(Sigalov et al., Advances in experimental medicine and biology,640:ix-xi, 2008). The consequences of combining several homologousreceptor components normally found in different receptors into a singleCAR has not been fully elucidated; however, energy andantigen-independent signaling have been observed with some designssuggesting that these receptors may not fully recapitulate the functionof the natural receptors upon which they are based (Brocker, Blood,96(5):1999-2001, 2000; Brocker et al., The Journal of experimentalmedicine. 181(5):1653-9, 1995; Milone et al., Molecular therapy: thejournal of the American Society of Gene Therapy, 17(8):1453-64, 2009).

The invention claimed herein describes CARs constructed upon a more“natural” split receptor design having greater potency in activating Tcells due to the naturally-selected interactions between the subunitswithin the receptor complex. The killer immunoglobulin-like receptor(KIR) system was chosen, which represents one of the simplest ITAM-basedreceptor systems, as the foundation for a CAR (Thielens et al., Currentopinion in immunology, 24(2):239-45, 2012). Although expressed bynatural killer cells where they contribute to their naturalcytotoxicity, KIR expression has also been observed in both CD4+ andCD8+ T cells (Moretta et al., Immunological reviews, 155:105-17, 1997;Falk et al., Human immunology; 61(12):1219-32, 2000; Remtoula et al.,Journal of immunology, 180(5):2767-71, 2008). Activating KIRs, such asKIR2DS2, possess a short cytoplasmic domain with no known endogenoussignaling capacity. Instead, these receptors form a non-covalent complexwith dimers of DAP12, an ITAM-containing adaptor molecule capable ofbinding Syk and Zap70 kinases in NK cells (Lanier et al., Nature,391(6668):703-7, 1998). In addition to stimulating cytotoxicity uponligand binding, KIRs have also been shown to exhibit costimulatoryeffects within T cells in the absence of DAP12 suggesting that thesemolecules might be able to provide both primary triggering activity andcostimulation in T cells (Snyder et al., Journal of immunology,173(6):3725-31, 2004).

A KIR-based CAR was constructed by splicing the mesothelin-specific SS1scFv onto the transmembrane and short cytoplasmic domain of theactivating KIR, KIR2DS2 (SS1-KIRS2) as illustrated schematically inFIGS. 1A and 1B (Hassan et al., Clinical cancer research: an officialjournal of the American Association for Cancer Research, 8(11):3520-6,2002). The ITAM-containing adaptor molecule, DAP12 is constitutivelyexpressed in natural killer (NK) cells, but it is only expressed in asubset of human T cells (Moretta et al.). Therefore, a bicistroniclentiviral vector encoding both the mesothelin-specific KIR-based CAR(SS1-KIRS2) and the DAP12 molecule separated by the Thoseaasigna virus2A (T2A) sequence was generated in order to achieve co-expression ofboth molecules (FIG. 2). Transduction of primary human T cells withSS1-KIRS2 and DAP12 bicistroinic lentivirus following anti-CD3 andanti-CD28 activation demonstrated robust surface expression of SS1-KIRS2that was comparable to the CD3ζ-based SS 1ζ CAR (FIGS. 6A and 6B).SS1-KIRS2/DAP12 co-transduced T cells expanded following polyclonalanti-CD3/anti-CD28 stimulation with kinetics that was comparable to thatobserved with mock transduced T cells or T cells transduced with amesothelin-specific CAR containing the CD3ζ cytoplasmic domain (data notshown). The cytotoxic activities of the KIR-based versus CD3ζ (SS1-z)CAR T cells was compared. SS1-KIRS2/DAP12-transduced T cells showedpotent cytotoxic activity towards K562 cells that express humanmesothelin (K-meso), but show no activity towards wild-type K562 (Kwt),similar in magnitude to the SS1ζ construct supporting the specificactivation of the SS1-KIRS2 receptor by the cognate mesothelin targetantigen (FIGS. 6A and 6B).

Since expression of KIR2DS2 has been described in T cells in the absenceof detectable DAP12 expression, the expression and function of theSS1-KIRS2 receptor with or without co-delivery of DAP12 was evaluated.Using a lentiviral vector that co-expressed DAP12 with the redfluorescent protein, dsRed (DAP12-dsRed) or a dsRed-expressing controlvector (dsRed), T cells were transduced with the lentiviral DAP12 orcontrol vector followed by transfection with in vitro transcribed RNAexpressing SS1-KIRS2. SS1-KIRS2 was expressed at the surface of T cellswithout the addition of DAP12; however, the surface expression ofSS1-KIRS2 increased by ˜1-log with the addition of DAP12 (FIG. 12A).Despite the expression of SS1-KIRS2 in the absence of DAP12 co-delivery,these T cells showed no appreciable cytotoxic activity in response tomesothelin-expressing target cells compared with T cells thatco-expressed SS1-KIRS2 and DAP12 (FIG. 12B). The data presented hereinsuggest that DAP12 is required for SS1-KIRS2 activity, but does notpreclude the possibility that the KIR domain might also provideadditional co-stimulatory activity independent of its association withDAP12 similarly to the natural KIR2DS2 receptor (Snyder et al.).

The non-covalent association of natural KIR2DS2 and DAP12 depends uponthe electrostatic interactions between an aspartic acid residue in theKIR transmembrane (TM) domain and a lysine residue in the DAP12 TMdomain (Feng et al., PLoS biology, 4(5):e142, 2006). Although theconfiguration of these ionizable amino acid residues in the TM domainsof TCR and CD3 subunits are thought to differ from the KIRs and DAP12,providing some specificity for the interactions, the possibility thatSS1-KIRS2 might be interacting with components of the CD3 complex inlieu of co-delivered DAP12 was investigated. Since the associationbetween the CD3 complex and TCR chains is required for TCR expression onthe cell surface, competition of the KIR for CD3 components would beexpected to interfere with normal TCR expression as previously observedwith expression of cloned TCRs (Varela-Rohena et al., Nature medicine,14(12):1390-5, 2008). Therefore, the effect of SS1-KIRS2 expression onthe expression of an endogenous TCR Vβ was evaluated. The frequency orintensity of TCR Vβ 14.3+ T cells were unaffected in T cells expressingthe SS1-KIRS2. The data presented herein suggests an absence of asignificant interaction between SS1-KIRS2 and members of the CD3 complex(FIG. 13).

Although cytotoxic activity is an important effector function for invivo anti-tumor activity of T cells, the ability of an antigen-receptorto trigger cytokine secretion and T cell proliferation are alsoimportant characteristics that generally correlate with robustanti-tumor activity in vivo. Therefore, the ability of T cellsexpressing SS1-KIRS2/DAP12 and CD3z-based CARs was compared withoutcostimulatory domains (SS1-ζ) or with CD28 or 4-1BB co-stimulatorydomains (SS1-28ζ and SS1-BBζ, respectively) to produce interferon-γ andIL-2 in response to mesothelin. The SS1-ζ construct produced the lowestlevels of both IFN-γ and IL-2 (FIG. 10, 11). Interferon-γ production washigher and similarly elevated in the T cells expressing SS1-KIRS2/DAP12or CD3ζ-based CARs bearing costimulatory domains (FIG. 10). T cells withthe CD3ζ-based CARs bearing costimulatory domains produced greaterquantities of IL-2 compared with T cells expressing SS1-ζ orSS1-KIRS2/DAP12 (FIG. 11). The SS1-KIRS2/DAP12 receptor was also apotent stimulator of T cell proliferation in response to cognate antigen(FIG. 14). Surprisingly, this proliferation was unaffected by theaddition of agonist antibody to CD28 (clone 9.3) suggesting thatadditional costimulatory signals are not required. The data presentedherein is consistent with a previously reported costimulatory functionof naturally expressed KIR2DS2 in human CD8+ T cell clones in theabsence of DAP12 (Snyder et al.). An alternative explanation is thatadditional receptors naturally expressed by T cells are also capable ofutilizing the co-delivered DAP12 further contributing to T cellactivation and proliferation.

That in vivo anti-tumor activity of T cells modified withmesothelin-specific TCR-ζ based CARs is significantly enhanced by theincorporation of costimulatory domains including CD28 and 4-1BB waspreviously demonstrated (Carpenito et al., Proceedings of the NationalAcademy of Sciences of the United States of America, 106(9):3360-5,2009); however the efficacy of a single intravenous injection of thesesecond or third generation CAR T cells into mice bearing establishedmesothelioma xenografts often failed to lead to complete tumorregression (Moon E K et al. Clin Cancer Res 2011; 17(14):4719-30; RieseM J et al. Cancer Res. 2013; 73(12):3566-77). Therefore, the activity ofSS1-KIRS2/DAP12-modified T cells in this resistant subcutaneous model ofmesothelioma was evaluated. The ability of SS1-KIRS2/DAP12-modified,DAP12-dsRed-modified T cells and SS1-28ζ T cells to kill EMMESO cells invitro was first tested. As shown in FIG. 15A, all three types ofCAR-modified T Cells showed similar in vitro killing efficacy, withminimal cytotoxicity induced by DAP12-dsRed-modified T cells. Micebearing large established EM-meso tumors were intravenously injectedwith 10⁷ T cells that were either mock-transduced T cells (mock) or Tcells transduced (at a similar level of transduction of ˜80%) withSS1-z, SS1-BBz, SS1-28z, or SS1-KIR-DAP12 and tumor growth was followed(FIG. 15A). In this model, the mock, SS1ζ, and the SS1BBζ T cells had nosignificant anti-tumor efficacy. The growth of the tumors wassignificantly (data not shown), but only modestly slowed by injection ofthe SS1-28ζ CAR T cells. In contrast, after a 10 day lag period, theSS1-KIRS2/DAP12-modified T cells induced marked tumor regressioninhibition of EM-meso tumor growth for up to 48 days. At this time, theanimals were sacrificed and the blood, spleens, and tumors analyzed.Flow cytometry was used to detect the presence of the human CD45+ cells(FIG. 15B). Only mice receiving the SS1-BBz CAR T cells had detectablehCD45+ cells in the blood and spleen. Within the tumors, no hCD45+ cellswere detected in mock T cell-treated mice and only a low percentage of Tcells were observed in SS1z-treated mice. In contrast, theSS1-KIRS2/DAP12, SS1-28z, and SS1-41BB CARs had hCD45+ cells thatcomprised 2-4% of the total viable cells with comparable frequenciesnoted for each group (FIG. 15B). The data presented herein demonstratethat the markedly increased efficacy of the SS1-KIRS2/DAP12 was not dueto larger frequency of T cells within the tumors.

To further explore the location of the T cells within the tumors,immunohistochemistry was performed. Staining showed CD8+ T cells andCD4+ T cells within the tumors within each group. Tumors from animalstreated with SS1-BBζ and SS1-28ζ CAR-modified T cells demonstratedparticularly dense T cell infiltrates; however, these infiltrates tendedto be within the periphery of the tumor suggesting that the SS1-BBζ andSS1-28ζ CAR-modified T cells might be limited in their ability totraffic and/or function with the tumor microenvironment compared withthe SS1-KIRS2/DAP12 CAR T cells.

Constitutive expression of DAP12 alone in murine T cells has beenreported to confer NK-like activity to these T cells with the ability tocontrol a solid tumor via NKG2D ligand-triggered effects (Teng et al.,The Journal of biological chemistry, 280(46):38235-41, 2005). AlthoughNKG2D has been reported to associate with DAP12 in mice, thisassociation appears to be absent in humans (Rosen et al. Journal ofimmunology, 173(4):2470-8, 2004). Despite the lack of in vitro cytolyticactivity by T cells expressing DAP12 only, a second in vivo experimentwas performed to compare T cells expressing DAP12 alone toSS1-KIRS2/DAP12 and to SS1-28z engineered T cells. Similar to the invitro results, DAP12-expressing T cells were unable to control EM-mesotumors in the absence of the mesothelin-specific SS1-KIR2S receptor(FIG. 15C). The data presented herein show an impressive anti-tumorresponse of the SS1-KIRS2/DAP12 T cells with frank tumor regression andalmost elimination of the tumors by Day 48.

T cell persistence in tumors has been shown to be an importantdeterminant of adoptive T cell transfer efficacy. However, the datapresented herein show that the enhanced effects of the KIR-CAR are notdue to increased numbers of T cells within the tumors; both the SS1-28zand SS1-41BBz constructs appear to persist at slightly higher numbersthan the KIR CARs. The location of the CAR T cells may be important,however. The staining data presented herein suggests thatSS1-KIRS2/DAP12 T cells may be more efficient in reaching the center ofthe tumors.

The mechanisms responsible for the markedly improved efficacy of theKIR-based CARs are under active investigation. Although both CD3 and KIRreceptor systems rely upon ITAM-based recruitment and activation ofdownstream signal, the nature of the ensuing ITAM-mediated signaling maynot be equivalent. Recently, a number of heterologous receptors with noknown mechanism for interaction with ITAM-containing receptors includingthe cytokine receptors for type I interferon and IL-3, the chemokinereceptors, CXCR4 and RANKL have been shown to depend uponITAM-containing receptor for signaling (Wang et al., Nature immunology,9(2): 186-93, 2008; Hida et al., Nature immunology, 10(2):214-22, 2009;Koga et al., Nature, 428(6984):758-63, 2004; Kumar et al., Immunity,25(2):213-24, 2006). It is therefore possible that the introduction ofDAP12 into T cells may alter a number of additional signals that mightbe relevant to T cell function within the complex tumormicroenvironment. The robust proliferation of T cells followingKIR-based CAR activation without additional co-stimulation might also bean important part of the enhanced efficacy of T cells modified withSS1-KIRS2 and DAP12. The clonal expansion of T cells following TCR andco-stimulatory receptor engagement requires tremendous syntheticdemands. Both CD28 and 4-1BB receptors are potent activators of the mTORpathway that is an important regulator of the metabolism required tosupport clonal expansion (Colombetti et al., Journal of immunology,176(5):2730-8, 2006; So et al., Frontiers in immunology, 4:139, 2013;Marelli-Berg et al. Immunology, 136(4):363-9, 2012). Interestingly,Berezhnoy et al. recently reported that interruption of mTOR using ansiRNA to raptor directed by a 4-1BB-specific RNA aptamer significantlyimproved the anti-tumor activity of T cells following therapeuticvaccination of tumor-bearing mice (Berezhnoy et al., The Journal ofclinical investigation. 124(1):188-97, 2014). Since costimulatorysignals such as CD28 and 4-1BB are normally regulated both temporallyand spatially during an immune response, this suggests that theunregulated costimulatory signals produced by the BBζ and 28ζ CARs,while critical for robust proliferation in response to CD3ζ CARtriggering, might have negative effects on T cell function in vivo,perhaps through persistent mTOR signaling.

In conclusion, the data presented herein demonstrate that thecombination of KIR-based CAR and DAP12 provides a highly effectivereceptor system for conferring artificial antigen specificity to Tcells. Despite relative equivalent in vitro activity, it has furtherbeen shown that this KIR-based CAR has much improved anti-tumor efficacycompared to CARs based on CD3ζ with one or more costimulatory domains inthe model tumor system utilized herein, perhaps due to increasedresistance to inactivation. Further exploration into the mechanisms ofthis increased efficacy and of chimeric receptor designs based uponother DAP12-associated ligand-binding receptors, as well as additionalnatural ITAM containing receptors systems such as FcRγ, will be pursued.

Example 7: A KIR-Based CAR can be Co-Expressed with a Natural InhibitoryKIR Permitting Regulation by HLA Expression on the Target CellsGeneration and Characterization of a K562-Meso Cell Line that Expressthe KIR2DL3 Ligand HLA-Cw

Material and Method:

Wild type K562 cells or a K562 line previously engineered to expressmesothelin (K562-meso) were transduced with a lentiviral vector encodingthe HLA-Cw3 allele. Cells were sorted for uniform expression ofmesothelin and HLA-Cw3 by fluorescence-activated cell sorting. HLA-Cw3expression was confirmed by flow cytometry following staining with theW6/32 anti-HLA A, B, C antibody conjugated to APC.

Result:

K562 cell lines expressing either mesothelin or HLA-Cw3 alone or incombination can be generated (FIG. 20).

Co-Expression of SS1-KIRS2 and KIR2DL3 in Primary Human T Cells

Material and Method:

Primary human T cells were stimulated with anti-CD3/28 microbeadsfollowed by transduction with either a bicistronic lentiviral vectorexpressing DAP12 and SS1-KIRS2 alone or in combination with a lentiviralvector expressing KIR2DL3 on day 1 following activation. The expressionof the SS1-KIRS2 CAR was assessed by flow cytometry using a biotinylatedgoat-anti-mouse F(ab)2 polyclonal antibody followed by SA-APC. KIR2DL3expression was determined using a KIR2D specific monoclonal antibody.

Result:

Primary human T cells expressing a mesothelin-specific KIR-based CARwith DAP12 (KIRS2) alone, KIR2DL3 alone or a combination of the tworeceptors can be generated (FIG. 21).

KIR2DL3 Coexpressed with a KIR CAR can Suppress Antigen SpecificCytotoxicity in the Presence of HLA-Cw on the Target Cells

Material and Method:

Primary human T cells were stimulated with anti-CD3/28 microbeadsfollowed by transduction with a bicistronic lentiviral vector expressingDAP12 and SS1-KIRS2. 5 μg of in vitro transcribed mRNA encoding KIR2DL3was introduced into the lentivirally-transduced T cells byelectroporation following 10 days of ex vivo expansion. These T cellpopulations were mixed with ⁵¹Cr-labeled K562 target cells (K562,K562-meso, K562-HLACw and K562-meso/HLACw) as indicated at varyingratios of effector T cells to target K562 cells (E:T ratio).Cytotoxicity was determined by measuring the fraction of ⁵¹Cr releasedinto the supernatant at 4 hours.

Result:

SS1-KIRS2/DAP12-expressing T cells were capable of killing target K562cells that express mesothelin regardless of HLA-Cw3 expression. Incontrast, T cells co-expressing the SS1-KIRS2/DAP12 receptor complex andthe inhibitory KIR, KIR2DL3 failed to exhibit robust cytotoxicityagainst K562 expressing mesothelin with HLA-Cw3; however, these cellsdemonstrated cytotoxic activity towards K562 cells expressing mesothelinalone that was comparable to SS1-KIRS2/DAP12-modified T cells. Theseresults demonstrate the ability of inhibitory KIR receptors to regulatethe functional activity of activating KIR-based CARs (FIG. 22).

Example 8: A KIR-Based CAR with CD19 Specificity can TriggerAntigen-Specific Target Cell Cytotoxicity In Vitro and In Vivo

A KIR-Based CAR with CD19 Specificity can Trigger Antigen-SpecificTarget Cell Cytotoxicity In Vitro

Material and Method:

Following anti-CD3/anti-CD28 bead activation, T cells were transducedwith a bicistronic lentiviral vector expressing DAP12 along with eithera CD19-specific KIR-based CAR in which the FMC63-derived scFv is fusedto full length KIR2DS2 (CD19-KIR2DS2) or a KIR-based CAR generated byfusing the FMC63 scFv to the transmembrane and cytoplasmic domain ofKIR2DS2 via a short linker [Gly]₄-Ser linker (SEQ ID NO: 66)(CD19-KIRS2). The transduced T cells were cultured until the end of thelog phase growth, and the expression of the CD19-specific KIR-based CARwas assessed by flow cytometry using a biotinylated goat-anti-mouseF(ab)₂ polyclonal antibody followed by SA-PE. ⁵¹Cr-labeled K562 targetcells with (K562-CD19) or without (K562-wt) CD19 expression were mixedat varying ratios with T cells to target cells (E:T ratio). Cytotoxicitywas determined by measuring the fraction of ⁵¹Cr released into thesupernatant at 4 hours. Control T cells that were either mock transduced(NTD) or transduced with a CD3ζ-based CAR specific to CD19 (CD19-z) werealso included as negative and positive controls, respectively.

Result:

Flow cytometric analysis demonstrates expression of the CD19-specificscFv on the surface of the T cells transduced with CD19-KIR2DS2,CD19-KIRS2 and CD19-z (FIG. 16A). T cells expressing DAP12 with eitherCD19-KIR2DS2 or CD19-KIRS2 were capable of killing target cells in anantigen-specific manner (FIG. 16B). Cytotoxicity exhibited by theKIR-based CAR-modified T cells was comparable to or higher than T cellsexpressing a CD19-specific CD3ζ-based CAR.

T Cells Transduced with CD19-KIRS2/DAP12 Induce Tumor Regression in aHuman Leukemia Xenograft

Material and Method:

NOD-SCID-γ^(−/−) (NSG) mice were engrafted intravenously by tail vein onday 0 with 1 million Nalm-6 CBG tumor cells, a leukemia cell lineexpressing CD19. In the experiment, T cells were stimulated withanti-CD3/anti-CD28 stimulator beads followed by lentiviral transductionon day 1 with a series of CD3-based CAR with or without a costimulatorydomain (CD19-z, CD19-BBz) or the CD19-specific KIR-based CARs,CD19-KIRS2 with DAP12 as indicated in the figure. Mock transduced Tcells (NTD) were used as a control. The T cells were expanded until theend of log-phase growth ex vivo and injected intravenously on day 5 postleukemia cell line injection with 2 million CAR T cells per mouse. Tumorburden was assessed via bioluminescent imaging. 5 animals were analyzedfor each T cell condition (FIGS. 17A and 17B).

Result:

In the in vivo experiment presented (FIGS. 17A and 17B), the NTD T cellshad no effect on tumor growth, while CD19z, CD19BBz□andCD19-KIRS2-transduced T cells exhibit various anti-tumor effects. Miceinfused with CD19z T cells showed a slight reduction in tumor burden butretained detectable levels of luminescence. In contrast, tumor cellluminescence in mice infused with either CD19BBz or CD19KIRS2 T cellsdropped to the lower limit of detection (FIG. 17B, dotted line) only 7days post T cell injection, exhibiting complete clearance outside of asmall reservoir of leukemia cells in the T cell-inaccessible tooth root.By day 15, tumor burden in the mock T cell group surpassed the endpoint(2×10¹⁰ photons/second) and were sacrificed, while luminescence in theCD19BBz and CD19KIRS2 groups remained at the lower limit of detection.

Example 9: A Camelid Single VHH Domain-Based CAR can be Expressed on a TCell Surface in Combination with a scFv-Based CAR without AppreciableReceptor Interaction

Material and Method:

Jurkat T cells expressing GFP under an NFAT-dependent promoter (NF-GFP)were transduced with either a mesothelin-specific activating CAR(SS1-CAR), CD19-specific activating (19-CAR) or a CAR generated using acamelid VHH domain specific to EGFR (VHH-CAR). Following transductionwith the activating CAR, the cells were then transduced with anadditional inhibitory CAR recognizing CD19 (19-PD1) to generate cellsco-expressing both the activating and inhibitory CAR (SS1+19PD1,19+19PD1 or VHH+19PD1). The transduced Jurkat T cells were co-culturedfor 24 hours with different cell lines that are either 1) devoid of alltarget antigens (K562), 2) express mesothelin (K-meso), CD19 (K-19) orEGFR (A431) only, 3) express a combination of EGFR and mesothelin(A431-mesothelin) or CD19 (A431-CD19) or 4) express a combination ofCD19 and mesothelin (K-19/meso). Additional conditions that includeeither no stimulator cells (no stim) or K562 with 1 ag/mL of OKT3 (OKT3)were also included as negative and positive controls for NFATactivation, respectively. GFP expression, as a marker of NFATactivation, was assessed by flow cytometry.

Result:

Camels and related species (e.g. Llama) naturally produce antibodiesthat have a single heavy-chain like variable domain. This domain, knownas a camelid VHH domain, has evolved to exist without pairing to a lightchain variable domain. FIG. 27A shows schematically the possibility thattwo heterologous scFv molecules can dissociate and re-associate with oneanother when displayed on the surface of a cell as demonstrated by theobserved disruption in scFv binding to cognate ligand during receptorco-expression (FIG. 25 and FIG. 26). FIG. 27B shows a schematicrepresentation of the expected reduced interaction between a scFv CARdisplayed on the surface of a cell in combination with a VHHdomain-based CAR. FIG. 28 demonstrates that coexpression of twoscFv-based CARs (SS1-z activating CAR and CD19-PD1 inhibitory CAR) onthe surface of a Jurkat leads to the inability of the activating CAR(SS1-z) to recognize its cognate ligand on the target cell and trigger Tcell activation despite the absence of the inhibitory receptor's ligand.This is consistent with the observed reduced ligand binding on thesurface (FIG. 25). In contrast, the coexpression of the same inhibitoryCAR (CD19-PD1) with a camelid VHH-based activating CAR (VHH-z) has noimpact on the ability of the VHH-based activating CAR to recognize itscognate EGFR ligand. These data support the model depicted in FIG. 27Bthat a VHH-based activating CAR can be expressed with an scFv-based CARwithout significant interaction between the receptors due to the reducedability of the scFv and VHH domains to interact.

Example 10: An NKp46-Based NCR CAR with Mesothelin Specificity TriggersAntigen Specific Cytotoxicity

Material and Method:

Following anti-CD3/anti-CD28 bead activation, T cells were transducedwith a bi-cistronic lentiviral vector expressing either DAP12 andSS1-KIRS2 (control), or FcεRγ and a mesothelin specific NKp46-based CAR(SS1-NKp46) or FcεRγ and a mesothelin-specific NKp46 CAR in which thenatural NKp46 extracellular domain was truncated (SS1-TNKp46). Theexpression of the mesothelian-specific CARs was assessed by flowcytometry using a biotinylated goat-anti-mouse F(ab)2 polyclonalantibody followed by SA-PE (FIGS. 18A-18B). The T cells were mixed with⁵¹Cr-labeled K562 target cells expressing mesothelin at varying ratiosof effector T cells to target K562 cells (E:T ratio). Cytotoxicity wasdetermined by measuring the fraction of ⁵¹Cr released into thesupernatant at 4 hours compared with spontaneous release.

Result:

Both the SS1-NKp46 and SS1-sNKp46 receptors exhibit surface expressionon T cells. SS1-TNKp46 transduced T cells show robust target cellcytolysis that is comparable to the KIR-based SS1-KIRS2 CAR. SS1-NKp46exhibited weaker cytotoxic activity that was evident only at higheffector to target cell ratios (FIGS. 18A-18B). These data demonstratethat an antigen-specific chimeric immunoreceptor for use in redirectingT cell cytolytic activity can be generated from natural cytotoxicityreceptors (NCRs) using a design similar to that used to create aKIR-based CAR.

Example 11: Interaction of scFv Domains

Material and Method:

In FIG. 24, Jurkat T cells were transduced with lentiviral vectorencoding a mesothelin-specific inhibitory KIR-based CAR (SS1-KIR2DL3).These transduced cells were then transduced with varying dilutions of alentiviral vector encoding a CD19-specific activating KIR-based CAR(CD19-KIR2DS2). These KIR-CARs are shown schematically in FIG. 23.Following transduction with both CARs, the frequency of cells withsurface expression of a CAR with an intact scFv capable of binding theirtarget ligand was assessed by flow cytometry following staining withboth a mesothelin-Fc fusion protein followed by a secondary anti-Fcantibody labeled with PE and an anti-CD19-specific (clone FMC63)anti-idiotype monoclonal antibody labeled with APC. In FIG. 25,anti-CD3/28-activated primary human T cells were transduced withdifferent lentiviral vectors encoding either a mesothelin-specificCD3z-based CAR bearing an mCherry fusion to the C-terminus (SS1z-mCh), aCD19-specific CAR with CD3z and 4-1BB cytoplasmic domain (19bbz) or acombination of both SS z-mCh and 19bbz. The expression of mCherry and afunctional SS 1 scFv was assessed by flow cytometry following stainingwith a mesothelin-Fc fusion protein followed by a secondary anti-Fcantibody labeled with FITC. In FIG. 26, anti-CD3/28-activated primaryhuman T cells were transduced with different lentiviral vectors encodingeither a mesothelin-specific CD3z-based CAR (SS1z), a CD19-specific CARbearing the FMC63 scFv (19bbz) or a CD19-specific CAR bearing the 21d4scFv (21d4bbz) or a CD19-specific CAR bearing the BL22 scFv (BL22bbz) inwhich the scFv was composed of either a heavy chain variable domain (VH)5′ to the light chain variable domain (VL) in the scFv (H2L) or the VLlocated 5′ to the VH (L2H). Following transduction with each of theCD19-specific CAR, the T cells were then co-transduced with SS1z. Thebinding of the SS1z to mesothelin and the surface expression of theanti-CD19 scFv was assessed by flow cytometry following staining with amesothelin-Fc fusion protein followed by a secondary anti-Fc antibodylabeled with FITC or biotinylated protein L followed bystreptavidin-conjugated APC.

Result:

FIG. 24 shows that coexpression of two intact, ligand-binding scFv-basedCARs (SS1-KIR2DL3 and CD19-KIR2DS2) on the cell surface is mutuallyexclusive. FIG. 26 demonstrates the loss of ligand binding occursdespite expression of the CAR in the cell as illustrated by the presenceof mCherry expressing cells with reduced mesothelin binding in cellsco-transduced with SS1z-mCh and 19bbz. FIG. 26 demonstrates that theinteraction between scFv leading to loss of scFv binding function can beobserved using different scFv-based CARs supporting the universal natureof this effect. These observations are consistent with the modeldepicted in FIG. 27A in which the variable domain of one scFv canundergo intermolecular pairing with a heterologous scFv-based chimericreceptor leading to loss of binding by the scFv within a single CAR.

Example 12: KIR-CAR Sequences

SS1 KIR2DS2 gene sequence (SEQ ID NO: 1)gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggtggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttattactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaagctagcACGCGTggtggcggaggttctggaggtgggggttcccagggggcctggccacatgagggagtccacagaaaaccttccctcctggcccacccaggtcccctggtgaaatcagaagagacagtcatcctgcaatgttggtcagatgtcaggtttgagcacttccttctgcacagagaggggaagtataaggacactttgcacctcattggagagcaccatgatggggtctccaaggccaacttctccatcggtcccatgatgcaagaccttgcagggacctacagatgctacggttctgttactcactccccctatcagttgtcagctcccagtgaccctctggacatcgtcatcacaggtctatatgagaaaccttctctctcagcccagccgggccccacggttttggcaggagagagcgtgaccttgtcctgcagctcccggagctcctatgacatgtaccatctatccagggagggggaggcccatgaacgtaggttctctgcagggcccaaggtcaacggaacattccaggccgactttcctctgggccctgccacccacggaggaacctacagatgcttcggctctttccgtgactctccctatgagtggtcaaactcgagtgacccactgcttgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagctccaaaaccggtaaccccagacacctgcatgttctgattgggacctcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgctgtaatggaccaagagcctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtgtcatacgcataaGtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctacgcttttgcgtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaa gatcagttggSS1 KIRS2 gene sequence (SEQ ID NO: 2)gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggtggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttattactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaagctagcggtggcggaggttctggaggtgggggttcctcacccactgaaccaagctccaaaaccggtaaccccagacacctgcatgttctgattgggacctcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgctgtaatggaccaagagcctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtgtcatacgcataaGtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctacgcttttgcgtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagtt ggSS1 KIR2DL3 gene sequence (SEQ ID NO: 3)gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggtggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttattactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaagCTAGCggtggcggaggttctggaggtgggggttccCAGGGGGCCTGGCCACATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCCCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCAGGTTTCAGCACTTCCTTCTGCACAGAGAAGGGAAGTTTAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTCCCATGATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAACGTAGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCACCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAACTCGAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAATAGTTGGCTTTCACCCACTGAACCAAGCTCCGAAACCGGTAACCCCAGACACCTGCATGTTCTGATTGGGACCTCAGTGGTCATCATCCTCTTCATCCTCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTGCAACAAAAAAAATGCTGTTGTAATGGACCAAGAGCCTGCAGGGAACAGAACAGTGAACAGGGAGGACTCTGATGAACAAGACCCTCAGGAGGTGACATATGCACAGTTGAATCACTGCGTTTTCACACAGAGAAAAATCACTCACCCTTCTCAGAGGCCCAAGACACCCCCAACAGATATCATCGTGTACACGGAACTTCCAAATGCTGAGCCCTGAGtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttattggaggcctacgcttttgcgtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagat gctgaagatcagttggCD19 KIR2DS2 construct sequence (SEQ ID NO: 4)gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatccGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAgctagcACGCGTggtggcggaggttctggaggtgggggttcccagggggcctggccacatgagggagtccacagaaaaccttccctcctggcccacccaggtcccctggtgaaatcagaagagacagtcatcctgcaatgttggtcagatgtcaggtttgagcacttccttctgcacagagaggggaagtataaggacactttgcacctcattggagagcaccatgatggggtctccaaggccaacttctccatcggtcccatgatgcaagaccttgcagggacctacagatgctacggttctgttactcactccccctatcagttgtcagctcccagtgaccctctggacatcgtcatcacaggtctatatgagaaaccttctctctcagcccagccgggccccacggttttggcaggagagagcgtgaccttgtcctgcagctcccggagctcctatgacatgtaccatctatccagggagggggaggcccatgaacgtaggttctctgcagggcccaaggtcaacggaacattccaggccgactttcctctgggccctgccacccacggaggaacctacagatgcttcggctctttccgtgactctccctatgagtggtcaaactcgagtgacccactgcttgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagctccaaaaccggtaaccccagacacctgcatgttctgattgggacctcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgctgtaatggaccaagagcctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtgtcatacgcataaGtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctacgcttttgcgtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagat gctgaagatcagttggCD19-PD1 chimeric CAR sequence (SEQ ID NO: 5)TGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAATACGACTCACTATAGGGAGACAAGCTTGCATGCCTGCAGGTCGACATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACGTCTCCTCAgctagcACGCGTggtggcggaggttctggaggtgggggttccaccctggtggttggtgtcgtgggcggcctgctgggcagcctggtgctgctagtctgggtcctggccgtcatctgctcccgggccgcacgagggacaataggagccaggcgcaccggccagcccctgaaggaggacccctcagccgtgcctgtgttctctgtggactatggggagctggatttccagtggcgagagaagaccccggagccccccgtgccctgtgtccctgagcagacggagtatgccaccattgtctttcctagcggaatgggcacctcatcccccgcccgcaggggctcagctgacggccctcggagtgcccagccactgaggcctgaggatggacactgctcttggcccctctgaGGATCCCCGGGTACCGAGCTCGAATTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACTAGTGGCGCC DAP12-T2A-SS1-KIRS2(SEQ ID NO: 6) 1464 bp DNA FEATURE Location DAP12    1..339 T2A sequence 352..408 SS1-scFv  481..1200 GS-linker 1207..1236KIR2DS2-derived sequence 1237..1464ATGGGGGGAC TTGAACCCTG CAGCAGGTTC CTGCTCCTGCCTCTCCTGCT GGCTGTAAGT GGTCTCCGTC CTGTCCAGGTCCAGGCCCAG AGCGATTGCA GTTGCTCTAC GGTGAGCCCGGGCGTGCTGG CAGGGATCGT GATGGGAGAC CTGGTGCTGACAGTGCTCAT TGCCCTGGCC GTGTACTTCC TGGGCCGGCTGGTCCCTCGG GGGCGAGGGG CTGCGGAGGC AGCGACCCGGAAACAGCGTA TCACTGAGAC CGAGTCGCCT TATCAGGAGCTCCAGGGTCA GAGGTCGGAT GTCTACAGCG ACCTCAACACACAGAGGCCG TATTACAAAG TCGAGGGCGG CGGAGAGGGCAGAGGAAGTC TTCTAACATG CGGTGACGTG GAGGAGAATCCCGGCCCTAG GATGGCCTTA CCAGTGACCG CCTTGCTCCTGCCGCTGGCC TTGCTGCTCC ACGCCGCCAG GCCGGGATCCCAGGTACAAC TGCAGCAGTC TGGGCCTGAG CTGGAGAAGCCTGGCGCTTC AGTGAAGATA TCCTGCAAGG CTTCTGGTTACTCATTCACT GGCTACACCA TGAACTGGGT GAAGCAGAGCCATGGAAAGA GCCTTGAGTG GATTGGACTT ATTACTCCTTACAATGGTGC TTCTAGCTAC AACCAGAAGT TCAGGGGCAAGGCCACATTA ACTGTAGACA AGTCATCCAG CACAGCCTACATGGACCTCC TCAGTCTGAC ATCTGAAGAC TCTGCAGTCTATTTCTGTGC AAGGGGGGGT TACGACGGGA GGGGTTTTGACTACTGGGGC CAAGGGACCA CGGTCACCGT CTCCTCAGGTGGAGGCGGTT CAGGCGGCGG TGGCTCTAGC GGTGGTGGATCGGACATCGA GCTCACTCAG TCTCCAGCAA TCATGTCTGCATCTCCAGGG GAGAAGGTCA CCATGACCTG CAGTGCCAGCTCAAGTGTAA GTTACATGCA CTGGTACCAG CAGAAGTCAGGCACCTCCCC CAAAAGATGG ATTTATGACA CATCCAAACTGGCTTCTGGA GTCCCAGGTC GCTTCAGTGG CAGTGGGTCTGGAAACTCTT ACTCTCTCAC AATCAGCAGC GTGGAGGCTGAAGATGATGC AACTTATTAC TGCCAGCAGT GGAGTAAGCACCCTCTCACG TACGGTGCTG GGACAAAGTT GGAAATCAAAGCTAGCGGTG GCGGAGGTTC TGGAGGTGGG GGTTCCTCACCCACTGAACC AAGCTCCAAA ACCGGTAACC CCAGACACCTGCATGTTCTG ATTGGGACCT CAGTGGTCAA AATCCCTTTCACCATCCTCC TCTTCTTTCT CCTTCATCGC TGGTGCTCCAACAAAAAAAA TGCTGCTGTA ATGGACCAAG AGCCTGCAGGGAACAGAACA GTGAACAGCG AGGATTCTGA TGAACAAGAC CATCAGGAGG TGTCATACGC ATAADAP12-T2A-SS1-KIRS2 (SEQ ID NO: 7) 488 aa Protein FEATURES LocationDAP12   1..113 T2A seq 118..136 Signal_peptide from CD8alpha  138..158SS1-scFv 161..400 GS-linker 403..412 KIR2DS2-derived seq 413..487Sequence MGGLEPCSRF LLLPLLLAVS GLRPVQVQAQ SDCSCSTVSPGVLAGIVMGD LVLTVLIALA VYFLGRLVPR GRGAAEAATRKQRITETESP YQELQGQRSD VYSDLNTQRP YYKVEGGGEGRGSLLTCGDV EENPGPRMAL PVTALLLPLA LLLHAARPGSQVQLQQSGPE LEKPGASVKI SCKASGYSFT GYTMNWVKQSHGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAYMDLLSLTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSGGGGSGGGGSS GGGSDIELTQ SPAIMSASPG EKVTMTCSASSSVSYMHWYQ QKSGTSPKRW IYDTSKLASG VPGRFSGSGSGNSYSLTISS VEAEDDATYY CQQWSKHPLT YGAGTKLEIKASGGGGSGGG GSSPTEPSSK TGNPRHLHVL IGTSVVKIPFTILLFFLLHR WCSNKKNAAV MDQEPAGNRT VNSEDSDEQD HQEVSYA FCERG-T2A-SS1-TNKp46(SEQ ID NO: 8) 1365 bp DNA FEATURES Location FCERG    1..258 T2A 271..327 Signal peptide from CD8alpha  331..393 SS1-scFv  400..1119GS-linker 1126..1155 NKp46-derived sequence 1156..1365 SequenceATGATTCCAG CAGTGGTCTT GCTCTTACTC CTTTTGGTTGAACAAGCAGC GGCCCTGGGA GAGCCTCAGC TCTGCTATATCCTGGATGCC ATCCTGTTTC TGTATGGAAT TGTCCTCACCCTCCTCTACT GCCGACTGAA GATCCAAGTG CGAAAGGCAGCTATAACCAG CTATGAGAAA TCAGATGGTG TTTACACGGGCCTGAGCACC AGGAACCAGG AGACTTACGA GACTCTGAAGCATGAGAAAC CACCACAGTC CGGAGGCGGC GGAGAGGGCAGAGGAAGTCT TCTAACATGC GGTGACGTGG AGGAGAATCCCGGCCCTAGG ATGGCCTTAC CAGTGACCGC CTTGCTCCTGCCGCTGGCCT TGCTGCTCCA CGCCGCCAGG CCGGGATCCCAGGTACAACT GCAGCAGTCT GGGCCTGAGC TGGAGAAGCCTGGCGCTTCA GTGAAGATAT CCTGCAAGGC TTCTGGTTACTCATTCACTG GCTACACCAT GAACTGGGTG AAGCAGAGCCATGGAAAGAG CCTTGAGTGG ATTGGACTTA TTACTCCTTACAATGGTGCT TCTAGCTACA ACCAGAAGTT CAGGGGCAAGGCCACATTAA CTGTAGACAA GTCATCCAGC ACAGCCTACATGGACCTCCT CAGTCTGACA TCTGAAGACT CTGCAGTCTATTTCTGTGCA AGGGGGGGTT ACGACGGGAG GGGTTTTGACTACTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAGGTGGAGGCGGTTC AGGCGGCGGT GGCTCTAGCG GTGGTGGATCGGACATCGAG CTCACTCAGT CTCCAGCAAT CATGTCTGCATCTCCAGGGG AGAAGGTCAC CATGACCTGC AGTGCCAGCTCAAGTGTAAG TTACATGCAC TGGTACCAGC AGAAGTCAGGCACCTCCCCC AAAAGATGGA TTTATGACAC ATCCAAACTGGCTTCTGGAG TCCCAGGTCG CTTCAGTGGC AGTGGGTCTGGAAACTCTTA CTCTCTCACA ATCAGCAGCG TGGAGGCTGAAGATGATGCA ACTTATTACT GCCAGCAGTG GAGTAAGCACCCTCTCACGT ACGGTGCTGG GACAAAGTTG GAAATCAAAGCTAGCGGTGG CGGAGGTTCT GGAGGTGGGG GTTCCTTAACCACAGAGACG GGACTCCAGA AAGACCATGC CCTCTGGGATCACACTGCCC AGAATCTCCT TCGGATGGGC CTGGCCTTTCTAGTCCTGGT GGCTCTAGTG TGGTTCCTGG TTGAAGACTGGCTCAGCAGG AAGAGGACTA GAGAGCGAGC CAGCAGAGCTTCCACTTGGG AAGGCAGGAG AAGGCTGAAC ACACAGACTC TTTGA FCERG-T2A-SS1-TNKp46(SEQ ID NO: 9) 455aa Protein FEATURES Location FCERG   1..86 T2A 91..109 Signal peptide from CD8alpha  111..131 SS1-scFv 134..373GS-liner 376..385 NKp46-derived sequence 386..454 SequenceMIPAVVLLLL LLVEQAAALG EPQLCYILDA ILFLYGIVLTLLYCRLKIQV RKAAITSYEK SDGVYTGLST RNQETYETLKHEKPPQSGGG GEGRGSLLTC GDVEENPGPR MALPVTALLLPLALLLHAAR PGSQVQLQQS GPELEKPGAS VKISCKASGYSFTGYTMNWV KQSHGKSLEW IGLITPYNGA SSYNQKFRGKATLTVDKSSS TAYMDLLSLT SEDSAVYFCA RGGYDGRGFDYWGQGTTVTV SSGGGGSGGG GSSGGGSDIE LTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSP KRWIYDTSKLASGVPGRFSG SGSGNSYSLT ISSVEAEDDA TYYCQQWSKHPLTYGAGTKL EIKASGGGGS GGGGSLTTET GLQKDHALWDHTAQNLLRMG LAFLVLVALV WFLVEDWLSR KRTRERASRA STWEGRRRLN TQTLDAP12-T2A-CD19-KIRS2 (SEQ ID NO: 10) 1470 bp DNA FEATURES Location DAP12   1..339 T2A sequence  352..408 CD19-scFv  481..481 GS-linker1213..1242 KIR2DS2-derived sequence 1243..1470 SequenceATGGGGGGAC TTGAACCCTG CAGCAGGTTC CTGCTCCTGCCTCTCCTGCT GGCTGTAAGT GGTCTCCGTC CTGTCCAGGTCCAGGCCCAG AGCGATTGCA GTTGCTCTAC GGTGAGCCCGGGCGTGCTGG CAGGGATCGT GATGGGAGAC CTGGTGCTGACAGTGCTCAT TGCCCTGGCC GTGTACTTCC TGGGCCGGCTGGTCCCTCGG GGGCGAGGGG CTGCGGAGGC AGCGACCCGGAAACAGCGTA TCACTGAGAC CGAGTCGCCT TATCAGGAGCTCCAGGGTCA GAGGTCGGAT GTCTACAGCG ACCTCAACACACAGAGGCCG TATTACAAAG TCGAGGGCGG CGGAGAGGGCAGAGGAAGTC TTCTAACATG CGGTGACGTG GAGGAGAATCCCGGCCCTAG GATGGCCTTA CCAGTGACCG CCTTGCTCCTGCCGCTGGCC TTGCTGCTCC ACGCCGCCAG GCCGGGATCCGACATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCTCTCTGGGAGA CAGAGTCACC ATCAGTTGCA GGGCAAGTCAGGACATTAGT AAATATTTAA ATTGGTATCA GCAGAAACCAGATGGAACTG TTAAACTCCT GATCTACCAT ACATCAAGATTACACTCAGG AGTCCCATCA AGGTTCAGTG GCAGTGGGTCTGGAACAGAT TATTCTCTCA CCATTAGCAA CCTGGAGCAAGAAGATATTG CCACTTACTT TTGCCAACAG GGTAATACGCTTCCGTACAC GTTCGGAGGG GGGACTAAGT TGGAAATAACAGGTGGCGGT GGCTCGGGCG GTGGTGGGTC GGGTGGCGGCGGATCTGAGG TGAAACTGCA GGAGTCAGGA CCTGGCCTGGTGGCGCCCTC ACAGAGCCTG TCCGTCACAT GCACTGTCTCAGGGGTCTCA TTACCCGACT ATGGTGTAAG CTGGATTCGCCAGCCTCCAC GAAAGGGTCT GGAGTGGCTG GGAGTAATATGGGGTAGTGA AACCACATAC TATAATTCAG CTCTCAAATCCAGACTGACC ATCATCAAGG ACAACTCCAA GAGCCAAGTTTTCTTAAAAA TGAACAGTCT GCAAACTGAT GACACAGCCATTTACTACTG TGCCAAACAT TATTACTACG GTGGTAGCTATGCTATGGAC TACTGGGGTC AAGGAACCTC AGTCACCGTCTCCTCAGCTA GCGGTGGCGG AGGTTCTGGA GGTGGGGGTTCCTCACCCAC TGAACCAAGC TCCAAAACCG GTAACCCCAGACACCTGCAT GTTCTGATTG GGACCTCAGT GGTCAAAATCCCTTTCACCA TCCTCCTCTT CTTTCTCCTT CATCGCTGGTGCTCCAACAA AAAAAATGCT GCTGTAATGG ACCAAGAGCCTGCAGGGAAC AGAACAGTGA ACAGCGAGGA TTCTGATGAACAAGACCATC AGGAGGTGTC ATACGCATAA DAP12-T2A-CD19-KIRS2 (SEQ ID NO: 11)489 aa Protein FEATURES Location DAP12   1..113 T2A seq 118..136Signal_peptide from CD8alpha  138..158 CD19-scFv 161..402 GS-linker405..414 KIR2DS2-derived seq 415..489 SequenceMGGLEPCSRF LLLPLLLAVS GLRPVQVQAQ SDCSCSTVSPGVLAGIVMGD LVLTVLIALA VYFLGRLVPR GRGAAEAATRKQRITETESP YQELQGQRSD VYSDLNTQRP YYKVEGGGEGRGSLLTCGDV EENPGPRMAL PVTALLLPLA LLLHAARPGSDIQMTQTTSS LSASLGDRVT ISCRASQDIS KYLNWYQQKPDGTVKLLIYH TSRLHSGVPS RFSGSGSGTD YSLTISNLEQEDIATYFCQQ GNTLPYTFGG GTKLEITGGG GSGGGGSGGGGSEVKLQESG PGLVAPSQSL SVTCTVSGVS LPDYGVSWIRQPPRKGLEWL GVIWGSETTY YNSALKSRLT IIKDNSKSQVFLKMNSLQTD DTAIYYCAKH YYYGGSYAMD YWGQGTSVTVSSASGGGGSG GGGSSPTEPS SKTGNPRHLH VLIGTSVVKIPFTILLFFLL HRWCSNKKNA AVMDQEPAGN RTVNSEDSDE QDHQEVSYA*4-1BB Intracellular domain (amino acid sequence) (SEQ ID NO: 12)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCD3 zeta domain (amino acid sequence) (SEQ ID NO: 13)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRCytoplasmic Domain of PD1 (SEQ ID NO: 14) Amino acids 192-288CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLCytoplasmic Domain of CTLA-4 (SEQ ID NO: 15) Amino acids 183-223AVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN IgG4H-hinge translation(SEQ ID NO: 49) 230 aa linear UNAESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISKAKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQEGNVFSCSVM HEALHNHYTQ KSLSLSLGKM IgDH-hinge translation (SEQ ID NO: 50)282 aa linear UNA RWPESPKAQA SSVPTAQPQA EGSLAKATTA PATTRNTGRGGEEKKKEKEK EEQEERETKT PECPSHTQPL GVYLLTPAVQDLWLRDKATF TCFVVGSDLK DAHLTWEVAG KVPTGGVEEGLLERHSNGSQ SQHSRLTLPR SLWNAGTSVT CTLNHPSLPPQRLMALREPA AQAPVKLSLN LLASSDPPEA ASWLLCEVSGFSPPNILLMW LEDQREVNTS GFAPARPPPQ PGSTTFWAWSVLRVPAPPSP QPATYTCVVS HEDSRTLLNA SRSLEVSYVT DH Human CD8 hinge(SEQ ID NO: 51) 43 aa linear UNA 10-FEB-2009TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFA

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-253. (canceled)
 254. A nucleic acid comprising a sequence encoding anactivating killer cell immunoglobulin-like receptor chimeric antigenreceptor (actKIR-CAR), wherein the actKIR-CAR comprises: anextra-cellular antigen binding domain from an antibody molecule or anon-antibody scaffold; an activating killer cell immunoglobulin-likereceptor (actKIR) transmembrane domain; and a cytoplasmic domain. 255.The nucleic acid of claim 254, wherein the actKIR transmembrane domaincan interact with the transmembrane domain of a DAP12 polypeptide. 256.The nucleic acid of claim 254, wherein the actKIR transmembrane domaincomprises a positively charged moiety.
 257. The nucleic acid of claim254, wherein the cytoplasmic domain is a KIR-cytoplasmic domain. 258.The nucleic acid of claim 254, wherein the antigen binding domain froman antibody molecule comprises a scFv.
 259. The nucleic acid of claim254, wherein the actKIR-CAR further comprises an extracellular hingedomain.
 260. The nucleic acid of claim 254, wherein the actKIR-CARfurther comprises an extracellular hinge domain that: (i) is other thana KIR hinge domain; (ii) is derived from a natural molecule; (iii)comprises a non-naturally occurring polypeptide sequence; (iv) is fromhuman CD8-alpha subunit; (v) is of less than 50, 20, or 10 amino acidsin length, or (vi) has fewer amino acids than a KIR2DS2 hinge domain.261. The nucleic acid of claim 254, wherein the actKIR-CAR comprises anactKIR cytoplasmic domain.
 262. The nucleic acid of claim 254, whereinthe actKIR-CAR can interact with and promote signaling from anImmunoreceptor Tyrosine-based Activation Motif (ITAM)-containingpolypeptide.
 263. The nucleic acid of claim 254, wherein the actKIR-CARcomprises a KIR D domain.
 264. The nucleic acid of claim 254, whereinthe actKIR-CAR comprises a KIR D1 domain or a KIR D2 domain.
 265. Thenucleic acid of claim 254, wherein the actKIR-CAR does not comprise aKIR D domain.
 266. The nucleic acid of claim 254, wherein the actKIR-CARcomprises a KIR2DS2 transmembrane domain or comprises a KIR2DS2cytoplasmic domain.
 267. The nucleic acid of claim 254, wherein theactKIR-CAR can interact with and promote signaling from a DAP12 or DAP10polypeptide.
 268. The nucleic acid of claim 254, wherein the actKIR-CARcan interact with and promote signaling from a DAP12 polypeptide. 269.The nucleic acid of claim 254, wherein the antigen binding domain froman antibody molecule comprises an immunoglobulin single domain antibody(sdAb).
 270. The nucleic acid of claim 254, wherein the antigen bindingdomain from an antibody molecule comprises a single light chain variabledomain (VL).
 271. The nucleic acid of claim 254, wherein the antigenbinding domain from an antibody molecule comprises a single heavy chainvariable domain (VH).
 272. The nucleic acid of claim 254, wherein theantigen binding domain from an antibody molecule comprises a nanobody.273. The nucleic acid of claim 254, wherein the antigen binding domainfrom an antibody molecule binds an antigen present on a cancer cell.274. The nucleic acid of claim 254, wherein the antigen binding domainfrom an antibody molecule binds an antigen present on a solid tumorcell.
 275. The nucleic acid of claim 254, wherein the antigen bindingdomain from an antibody molecule binds an antigen present on ahematological tumor cell.
 276. The nucleic acid of claim 254, whereinthe antigen binding domain from an antibody molecule binds CD19,mesothelin, CD123, or BCMA.